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

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

p62/SQTM1 was initially characterized as the mediator of NFκB signaling and is now known as the signaling hub for a variety of cellular events including oxidative stress response and amino acid sensing [37]. As discussed previously, p62 is a signaling adaptor which links autophagic substrates to autophagic machineries. During autophagy, the degradation of p62 serves as a marker for functional autophagic activities. Thus, in a Western blot, the accumulation of p62 protein can be indicative of autophagy inhibition. While the principle of p62 assays seems straightforward, caution has to be taken during the interpretation by taking into account the regulation of p62 concentration at the transcriptional and post-translational stages. Taking autophagy out of the context, compilations of reports and studies demonstrate the link between high cytoplasmic but low nuclear expression levels with high tumor aggression and poor prognosis. The cancer types studied include endometrial cancer, oral squamous cell carcinoma, NSCLC, and epithelial ovarian cancer. Specifically, in breast cancer, high cytoplasmic expression levels of p62 have been shown to correlate with cancer grade, metastatic status, and reduction in five-year survival. Additionally, it was also significantly associated to high levels of epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor-2/3/4 (HER2/3/4) expression. Poor prognosis due to high levels of cytoplasmic p62 expression also holds true in triple-negative breast cancer [38]. In general, the studies available to date indicate high cytoplasmic p62 expression as a marker for poor prognosis. More studies will have to be

done on nuclear-localized p62 across cancer types to understand its prognostic values.

The Beclin-1/VPS34 complex is also one of the most popularly studied autophagy-related genes as it is the central coordinator of autophagy. Normally, Beclin-1 inhibits the anti-apoptotic Bcl-2 proteins; thus, the reduction of Beclin-1 expression can lead to enhanced anti-apoptotic pathways, consequently translating to poor prognosis. Overexpression on the other hand induces tumor hypoxia and acidity, which is likely to promote autophagy. Such events may increase aggressive tumor behavior [39]. Beclin-1 is a favorable prognostic marker in several cancers including lymphoma, lung, breast, and gastric cancer. In the context of colorectal cancer, the prognostic values can be split between poor and favorable. Poor prognosis in colorectal cancer patients was demonstrated in a study by Han et al. [40], and this finding was supported by additional studies by other researchers. The contradiction between colorectal cancer and other cancer types was addressed by Han et al. when he found that high expression level was associated to poor survival in patients undergoing chemotherapy, whereas patients without chemotherapy showed higher overall survival. Since most chemotherapeutic drugs actively induce

carcinoma [36], and clear cell ovarian carcinoma [30, 37].

**3.2. p62/SQSTM1**

80 Cell Culture

**3.3. Beclin-1/VPS34**

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.

## **3.5. ATG4B**

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

#### **3.6. GABARAP**

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].
