**3.1. LC3B and LC3A**

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

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].

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].

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,

that DAPk has antimetastasis and tumor suppressor properties [20].

**2.2. Autophagy as tumor promoter**

78 Cell Culture

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

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.

LC3A on the other hand is another autophagy marker that is being studied, albeit not as extensive as LC3B. IHC staining of LC3A can be defined into three distinct staining patterns: juxtanuclear staining, staining of "stone-like" structures (SLS), and diffuse cytoplasmic staining [32]. All three patterns translate to different prognostic values. For example, a high staining pattern of juxtanuclear LC3A in colorectal [33] and breast cancers [32] correlates with good prognosis; however, the opposite is true if it was SLS. Accumulation of SLS is also linked to poor prognosis in other cancer types including gastric [34], NSCLC [35], hepatocellular carcinoma [36], and clear cell ovarian carcinoma [30, 37].

autophagy [41], it is possible that the increased autophagic rate caused by high expression levels promoted resistance to chemotherapy. As with p62, expression levels of Beclin-1 in different histologic and genetic subtypes of breast cancer must be examined. To date, Beclin-1 expression was shown to be significantly correlated to estrogen receptor (ER) negativity. Expression levels vary between subtypes, with maximum expression found in triple-negative breast cancer [42].

Cell-Based Assays for Evaluation of Autophagy in Cancers

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

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ULK-1 and ULK-2 are serine/threonine kinases that are the most upstream components of the autophagy pathway and as such became attractive drug targets. While ULK-1/ULK-2 small molecule inhibitors are being developed, the data on the prognostic value of ULK-1/ULK-2 in different cancers is contradictory and limited. In breast cancer [40] and gastric cancer [43], high expression of ULK-1 appears to associate to good patient prognosis. This contradicts the findings in colorectal cancer [44]. A separate study in colorectal cancer did not demonstrate any correlation to survivability, even after taking into account the status of KRAS. However, they found that high expression levels were linked to lymph node metastasis. High levels of ULK-1 expression were also translated to adverse prognosis in esophageal squamous cell carcinoma [45] and nasopharyngeal carcinoma [46]. As for ULK-2, high expression levels were reported in prostate cancer tissue compared to adjacent healthy prostate tissue, but no correlation to prognosis was possible from this study [47]. With these contradictions in mind, additional studies with bigger patient cohorts will be required to further understand the prognostic values of ULK-1 and ULK-2 in cancer.

ATG4B is a cysteine protease that plays a central role in autophagy. While it is one of the autophagy proteins that is being targeted as a potential therapeutic protein, not much is known about its prognostic value. Some studies showed elevated expression levels in chronic myeloid leukemia, colorectal, and lung cancer; however, there is no mention of its prognostic

GABARAP is a part of the ATG8 protein homologs that consist of the subfamily LC3s and GABARAPs. While LC3 has been widely studied as the marker for autophagy, little is known about the GABARAP. GABARAP subfamily consists of GABARAP, GABARAPL1, and GABARAPL2 (GATE16) [48]. While LC3B is responsible in the elongation of autophagosome, Joachim et al. suggests that GABARAP lipidation onto the phagosome keeps the ULK-1 in an activated state until the dissociation of ULK-1 complex and the closing of the phagosome [49]. GABARAPL1 has been implicated in the formation of tumor in mice exposed to genotoxic DMBA [50]. Knockout GABARAPL1 mice showed a reduced expression of TGF-β1, which acts as a suppressor of T helper 1 cells (TH1). The reduced suppression on TH1 results in increased expression of cytokines IL-2 and IFN-γ, which induces an immune response during the exposure of DMBA on the knockout mice. Salah et al. also suggest that the cytokines promote the expression of Xaf1, which inhibits Xiap, a negative regulator of apoptosis, thus promoting cell death and preventing formation of cancerous cells. On the other hand, Berthier et al. found that GABARAPL1 inhibits the growth of breast cancer cell line, MCF-7, and the

expression of *GAPARAPL1* gene is downregulated in breast cancer tissues [51].

value in cancers, including members of the ATG4 family [30].

**3.4. ULK-1/ULK-2**

**3.5. ATG4B**

**3.6. GABARAP**
