**3.2. p62/SQSTM1**

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.

#### **3.3. Beclin-1/VPS34**

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 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].
