4. SQSTM1/p62 Is Related to Autophagy and Apoptosis in CRC Cells
SQSTM1/p62 is an autophagy receptor involved in optimizing the autophagosome formation process. After the fusion of autophagosomes with lysosomes, the autophagosome content, as well as SQSTM1/p62, is degraded
[26]. SQSTM1/p62 plays specific and indispensable roles in selective autophagy and SQSTM1/p62 protein levels have been used as an indicator of autophagic flux
[27]. The pivotal roles of SQSTM1/p62 in ingested protein catabolism through autophagy and lysosomal targeting have been well elucidated in the literature
[26][27][28]. Although the role of SQSTM1/p62 in solid tumors is still debated, evidence has shown that SQSTM1/p62 is upregulated in different cancers and promotes tumor growth. Moreover, it has been assessed that SQSTM1/p62, as a tumor oncogene, is frequently abnormally upregulated and engaged in the acquired malignancy of gastrointestinal tumors, such as CRC
[29].
Recently, Mukherjee et al. stated that, in HCT116 colorectal cells, SQSTM1/p62 is a mutp53 interactor, which associates selectively with the DNA contact mutant p53R273H but not with the structural mutant p53R175H. They further assess that the interaction with SQSTM1/p62 is needed for the ability of p53R273H to trigger cancer cell migration and invasion. The scholars assumed that this acquired ability is due to the involvement of the mutp53-p62 axis in directing the ubiquitin-dependent proteasome degradation of connexin 43 and cell junction proteins
[30]. Nevertheless, the autophagic process can be repressed by SQSTM1/p62 inhibition, leading to the arrest of cancer cell growth and the inhibition of cancer development
[31]. On the other hand, literature has reported that SQSTM1/p62 expression in CRC cells was suppressed by the β-catenin/transcription factor (TCF)4 complex, which blocked phagocytic ingestion, but under stress conditions induced by nutrient depletion, SQSTM1/p62 was markedly raised, as β-catenin was immediately degraded by LC3 binding to the LC3-interacting region (LIR) of β-catenin
[31].
5. NF-κB as a Potential Matchmaker between Autophagy and Apoptosis
Studies on CRC have reported that the NF-kB signaling pathway promotes tumor initiation and contributes to cancer cell metastasis formation and epithelial to mesenchymal transition
[32][33][34]. In the literature, there is a plethora of papers that confirm the role of NF-kB in regulating the apoptotic process. In fact, the molecular mechanisms by which NF-kB exerts its regulatory action are well known, for instance: NF-κB signaling prevents apoptosis by up-regulating anti-apoptotic genes expression such as B-cell lymphoma-extra-large (Bcl-xL), the Bcl-2-related gene (A1/BFL1), cellular inhibitors of apoptosis proteins (cIAPs) and caspase-8/FAS-associated death domain-like IL-1beta-converting enzyme inhibitory protein (c-FLIP)
[33]. Over the past few years, some very interesting studies have highlighted a correlation between NF-kB, autophagy and apoptotic processes. A study showed that metformin had an antiproliferative effect related to changes in the expression of nuclear factor E2-related factor (NRF-2)/NF-κB pathways on human colon cancer cells (HT-29) in a dose- and time-dependent manner, and exerted growth inhibitory effects by increasing both apoptosis and autophagy
[35].
Recently, the autophagy-regulated cytotoxic effect of green synthesized silver nanoparticles (Brassica Ag-NPs) against human epithelial colorectal adenocarcinoma cells was demonstrated. The scholars found that Brassica Ag-NPs induced NF-κB mediated autophagy in Caco-2 cells. The decreased expression of NFκB was associated with an increased expression of inhibitor of NF-κB (IκB)-kinase, which is involved in autophagic process initiation. Moreover, the activity of p53 and light chain 3 (LC3) II greatly accelerated autophagosome formation, and the inhibition of Akt and mammalian target of rapamycin (mTOR) was evident. In conclusion, the scholars stated that, in the event of excessive activation of the autophagic process, after the complete depletion of nutrients, this process stalled, leading to the accumulation of aberrant metabolic intermediates that can trigger apoptotic cell death, which subsequently resulted in necrosis [36].
6. Oxidative Stress and Modulation of Autophagy and Apoptosis
Oxidative stress has been implicated in the pathophysiology of cancer: high levels of intra-cytoplasmatic reactive oxygen species (ROS), generated by accelerate aerobic glycolysis followed by “selfish” metabolic reprogramming (the Warburg effect), increase oncogene activity, the activation of growth factor dependent pathways or the presence of an increase in the pool of oxidizing enzymes, leads to genetic instability
[37][38]. Aerobic glycolysis or the Warburg effect is a hallmark of metabolic phenotypes of cancer; in fact, cancer cells, including CRC cells, present altered glucose metabolism and are characterized by an enhanced uptake of glucose and an increased conversion of glucose to lactate
[39]. In the past few years, many studies have focused on the importance of the role played by ROS in the crosstalk between autophagy and apoptosis of colon cancer cells. ROS has a negative feedback action on the autophagic process: ROS can promote autophagy, and at the same time, autophagy reduces ROS production by removing damaged mitochondria, endoplasmic reticulum and other materials that contribute to ROS generation
[40][41]. On the other hand, ROS can trigger DNA damage, initiating the apoptotic process pathway in colon cancer cells
[42][43]. The most common method by which ROS delete transformed cells is the activation of programmed cell death, which is completed by an extrinsic or an intrinsic pathway; both pathways culminate in caspase-induced final cell demise with the formation of apoptotic bodies that are removed by adjacent phagocytes (
Figure 24)
[44].
Figure 24. Oxidative stress promotes the activation of programmed cell death initiated by intrinsic apoptotic signaling in the mitochondria or extrinsic apoptotic signaling by death receptor pathways in CRC cells. [Apaf 1, Apoptotic protease activating factor 1; BAD, Bcl-2-Associated agonist of cell death; BAX, Bcl-2-associated X protein; Bcl-2, B-cell lymphoma-2; BID, BH3-interacting domain death agonist; cyto c, cytochrome c; tBID, C-terminal fragment termed truncated Bid].
In the past few years, several interesting articles have been published concerning the inducing effect on both apoptosis and autophagy of drugs, mostly of natural origin, through regulatory pathways mediated by oxidative stress
[45][46][47][48][49]. In this regard, a study investigated how β-elemene, extracted from the genus Curcuma, affected cells in vitro and in vivo. The results reported that after β-elemene treatment, CRC cells exhibited apoptotic bodies and increased levels of cleaved-caspase-3/9 and PARP proteins. The in vivo experiments conducted on mouse-xenograft models inoculated with mouse colon carcinoma HT-29 cells presented the same expression pattern of apoptotic markers. Moreover, β-elemene promoted the formation of intracellular acidic vesicle organelles (autophagy lysosomes) and boosted the expression of autophagic markers such as LC3B and SQSTM1/p62 in human CRC cells.
7. Role of Non-Coding RNA in Enhancing or Inhibiting Autophagy and Apoptosis
With the deepening of the knowledge about the interaction between autophagy and apoptosis in CRC, researchers gradually begin to pay attention to the role of non-coding-RNA in this interplay. microRNAs (miRNAs) are a group of evolutionarily conserved small RNAs, endogenous, non-coding, single-stranded RNAs that regulate the expression of their target genes through mRNA degradation and serve a negative role at post-transcriptional levels. miRNAs have been found to influence many cellular biological processes including apoptosis, cell cycle distribution and autophagy in CRC
[50][51][52][53]. In detail, as regards autophagy, miRNAs may play a dual, and therefore conflicting, role in tumor cell proliferation through modulation of the autophagic process. While miR-211 promote CRC proliferation by inhibiting autophagy and targeting tumor protein 53-induced nuclear protein 1 (TP53INP1)
[54], on the other hand, miR-30d suppresses cell proliferation in CRC by inhibiting autophagy. The scholars demonstrated that miR-30d inhibited autophagy and promoted apoptosis of colon cancer cells by directly targeting ATG5, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit beta (PIK3CB) and beclin 1 (BECN1); the inhibition of proliferation was due to negative regulation of cell autophagy and promotion of cell death
[55].
8. Pathways Implicated in Regulation of Apoptosis and Autophagy and Possible Therapeutic Targets
The most important pathway involved in the induction and regulation of apoptosis and autophagy in CRC is the Wnt pathway and its component components, such as β-catenin, disheveled (Dvl), adenomatous polyposis coli (APC) and axin, that are activated along with the Wnt signaling cascade during cancer development
[56]. Wnt signal transduction is typically divided into classical and non-classical pathways. The classical pathway is involved in cell survival, proliferation, apoptosis and autophagy, while the non-classical pathway regulates cell polarity and migration. The Wnt/β-catenin signaling and autophagy pathways play important roles in tissue homeostasis and tumorigenesis. A variety of studies, reporting experimental design of the genomic manipulation of β-catenin expression levels in vitro and in vivo, show that β-catenin inhibits autophagosome formation and directly blocks SQSTM1/p62 via T cell-specific DNA-binding protein (TCF4)
[57]. During starvation, β-catenin is selectively degraded through the formation of β-catenin-LC3 complexes, which avoid β-catenin/T cell-specific DNA-binding protein (TCF) mediated transcription and proliferation in order to better adapt to metabolic stress conditions. The formation of the β-catenin-LC3 complex is mediated by the W/YXXI/L motif and the LC3 interaction region (LIR) in β-catenin. Therefore, as previously described, Wnt/β-catenin inhibits autophagy and SQSTM1/p62 expression, while β-catenin itself is targeted for autophagic degradation
[58]. Autophagy negatively controls Wnt signaling by promoting Dvl degradation. Von Hippel–Lindau protein-mediated ubiquitination is necessary for the binding of Dvl2 to SQSTM1/p62, leading to the formation of Dvl2 aggregates under conditions of nutrient deprivation and LC3-mediated autophagosome recruitment. Finally, the ubiquitylated Dvl2 aggregates are degraded through the autophagy-lysosome pathway
[59]. Interestingly, an inverse correlation between Dvl expression and autophagy was found in late stages of CRC development, supporting the hypothesis that autophagy may contribute to the aberrant promotion of Wnt signaling during tumorigenesis
[56].
9. Conclusions
While the antagonistic effects of autophagy and apoptosis occur in an adverse environment characterized by deprivation of oxygen and nutrients leading to the formation and development of CRC, the effects of promotion and collaboration usually involve an auxiliary role of autophagy compared to apoptosis in colon cancer cells. In an overwhelming majority of cases, studies examining the mechanistic role of autophagy and apoptosis on CRC tumorigenesis used xenograft mice models and CRC cell lines. Moreover, many of these studies have targeted autophagy and apoptosis transiently through genetic manipulations or the use of drugs, which can influence the study in various ways. From our point of view, it would be necessary for preclinical research to focus on the improvement of gene-editing techniques, which are necessary to delineate the mechanistic role of autophagy and apoptosis in CRC. The results obtained at this stage would be of great use in clinical trials in patients to test the response to immune-checkpoint inhibitors. In addition, it is necessary to focus attention on ongoing clinical trials that are using apoptosis or autophagy inhibitors in combination with chemotherapeutics or drugs. These studies aim to capture the positive results of using autophagy as a process of inducing programmed cell death in cancer cells. Such autophagic manipulation can lead to promising effects in the treatment of CRC if it will attempt to solve the autophagy paradox that migrates from an anti-cancer to a pro-cancer mechanism, so treatments need to be highly specific for the given setting.