**2. Role of autophagy in cancers**

### **2.1. Autophagy as tumor suppressor**

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 neuroblastoma degeneration undergoing autophagic cell death showed high expressions of RAS [23]. Similarly, this phenomenon was also observed in neuroblastoma and HT-29 colon cancer cell lines in vitro [20]. DAPk was initially identified as a cell death-promoting protein before its role in mediating autophagy and tumor suppression was characterized [24]. Like other cases of tumor suppressors, DAPk expression was found to be reduced in several cancer cell lines including but not limited to bladder, breast, renal, lung, ovarian, cervix, colon, head and neck, prostate, and brain cancers. The reintroduction of DAPk into highly metastatic cell lines ameliorated metastasis and tumorigenesis and improved cell death. These data suggest that DAPk has antimetastasis and tumor suppressor properties [20].

#### **2.2. Autophagy as tumor promoter**

Contrary to what was discussed above, cancer cells also rely on autophagy for survival and in many cases can be more dependent than normal cells. This is possibly due to the heavy reliance of tumors on nutrient supplies for the maintenance of rapid cell proliferation. In tumors inflicted with hypoxia, autophagy is essential for tumor survival [25]. In RAS-transformed cancer cells, autophagy was shown to be upregulated, and this promotes their survival, growth, invasion, and metastasis. The upregulation of autophagy in RAS-driven cancers has also been reported to ameliorate mitochondrial metabolic defects and the resulting sensitivity to stress. The genetic mechanism on how autophagy dependency arises is still at its infant stage [15].

drug development wecules targeting autophagy proteins. An example of lysosomal inhibitors will be the chloroquine (CQ) and hydroxychloroquine (HCQ), both of which are the main characters in clinical trials. The derivative of chloroquine Lys05 is currently going through optimization for clinical trials [30]. LC3B and p62 are common autophagy markers generally used in some assays to assess autophagy turnover, and these two can be useful to monitor the effectiveness of autophagy treatment. Potential targets for autophagy proteins to be discussed in the next section include LC3A, LC3B, p62, ULK, ATG4, ATG7, and Beclin-1. **Figure 2** summarizes the tumor promoting and suppressing roles of autophagy in cancers as well as their

Cell-Based Assays for Evaluation of Autophagy in Cancers

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LC3B is an extensively studied autophagy-related protein and is the shortened name for microtubule-associated protein 1 light chain 3B. Together with p62, LC3B has been used as an autophagy marker in several laboratory assays to measure autophagy flux. While in general a high expression of LC3B in cancer patients represents a bad overall prognosis, there are exceptions. This is especially apparent in breast cancer, where the genetic disposition of the cancer subtype will determine the prognostic values of LC3B expression. In the context of colorectal cancer, poor prognosis with high LC3B expression was observed only in KRASmutated samples but showed no changes in KRAS-WT specimens [31]. Interestingly, a high expression of LC3B was found to be in favor of the patient's prognosis in NSCLC [31]. With these different findings, LC3B cannot be used as a general prognostic biomarker across all cancer types or even within cancer subgroups. It will be important to study and identify its

prognostic value with attention to different cancer types and subtypes.

therapeutic potentials.

**3.1. LC3B and LC3A**

**3. Autophagy markers**

**Figure 2.** The role of autophagy in cancers and its therapeutic value.

In a study specific on the role of autophagy in GEM models for RAS-driven non-small-cell lung cancer (NSCLC), the essential autophagy gene ATG7 was deleted together with mutant KRAS in tumor cells. They observed that the absence of ATG7 causes an aberrant accumulation of defective mitochondria, and this leads to the accelerated activation of p53, cell growth arrest, and cell death. ATG7 loss also results in reduced tumor burden by facilitating the conversion of adenomas and carcinomas to a rare form of benign neoplasms called oncocytomas, characterized by the accumulation of nonfunctional mitochondria [26]. While it may seem beneficial to reduce the expression of ATG7 in cancer cells, there is no extension to the life-span of the mice as they die of pneumonia, possibly triggered by inflammation caused by autophagy defects [15]. The deletion of an alternative autophagy gene ATG5 in the same setting also resulted in a similar reduction of tumorigenesis, suggesting that tumorigenesis is mediated by autophagy itself and not just by ATG7 alone. Similarly, the activation of autophagy in GEM models of RASdriven pancreatic cancer also suppresses p53 activation [27]. In a separate study, it was found that the allelic loss of BECN1 promotes the activation of tumor suppressor p53, and this in turns reduced tumorigenesis [28]. However, large-scale genomic analysis to date has not been successful in identifying recurrent mutations in essential autophagy genes such as BECN1 [29].

#### **2.3. Potential targets and biomarkers in cancers**

Oftentimes, the pathways of important cellular processes such as apoptosis and inflammation that can be linked to diseases are studied extensively in hopes of searching for biomarkers or potential drug targets. Autophagy is no exception, as its role in cancer has been demonstrated in the previously mentioned studies; several groups are investigating their predictive and prognostic values. As of 2017, most of the characterized autophagy-related protein markers are prognostic in nature but are not approved for use in clinical settings. To date,

**Figure 2.** The role of autophagy in cancers and its therapeutic value.

drug development wecules targeting autophagy proteins. An example of lysosomal inhibitors will be the chloroquine (CQ) and hydroxychloroquine (HCQ), both of which are the main characters in clinical trials. The derivative of chloroquine Lys05 is currently going through optimization for clinical trials [30]. LC3B and p62 are common autophagy markers generally used in some assays to assess autophagy turnover, and these two can be useful to monitor the effectiveness of autophagy treatment. Potential targets for autophagy proteins to be discussed in the next section include LC3A, LC3B, p62, ULK, ATG4, ATG7, and Beclin-1. **Figure 2** summarizes the tumor promoting and suppressing roles of autophagy in cancers as well as their therapeutic potentials.
