Colorectal cancer (CRC) remains a major life-threatening malignancy. Apoptosis and autophagy are two processes that share common signaling pathways, are linked by functional relationships and have similar protein components. During the development of cancer, the two processes can trigger simultaneously in the same cell, causing, in some cases, an inhibition of autophagy by apoptosis or apoptosis by autophagy. Malignant cells that have accumulated genetic alterations can take advantage of any alterations in the apoptotic process and as a result, progress easily in the cancerous transformation. Autophagy often plays a suppressive role during the initial stages of carcinogenicity, while in the later stages of cancer development it can play a promoting role. It is extremely important to determine the regulation of this duality of autophagy in the development of CRC and to identify the molecules involved, as well as the signals and the mechanisms behind it. All the reported experimental results indicate that, 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.
CRC remains a major life-threatening malignancy, despite numerous therapeutic and screening attempts 
. During CRC development, programmed cell death plays a crucial role in the transformation process of benign polyps into adenomas and ultimately adenocarcinoma 
. During the multistep process of human colorectal carcinogenesis, the key proteins regulating the programmed death of mucosal cells undergo fundamental expression changes. The cells of the normal mucosa evolve towards a neoplastic condition and acquire new characteristics and biological capabilities, among which the ability to escape cell death is one of the most important 
. The multistep process of human carcinogenesis requires that cancer cells acquire the features enabling them to become tumorigenic and ultimately malignant. The apoptotic machinery is very complex and diversified and is composed of both upstream regulators and downstream effector components 
. The existing balance between pro and anti-apoptotic factors belonging to the B-cell lymphoma-2 (Bcl-2 family) of regulatory proteins is the basis of the control of the “apoptotic trigger” that conveys signals between regulators and effectors. Moreover, several abnormality sensors that play key roles in tumor development have been identified, among which a DNA damage sensor that functions via the tumor suppressor p53 (TP53) is most notable 
. Apoptosis and autophagy are two processes that share common signaling pathways, are linked by functional relationships and have similar protein components. During the development of cancer, the two processes can trigger simultaneously in the same cell, causing, in some cases, an inhibition of autophagy by apoptosis or apoptosis by autophagy 
. In particular, several genes and proteins are part of the complex mechanism of autophagy. Recent evidence has demonstrated that autophagy plays a decisive role in all stages of human tumorigenesis, by leading initiation and contributing to progression and metastatic ability in tumors. The signal pathways that are activated in the autophagic process during carcinogenesis are not yet defined and appear to be dependent on the tumor microenvironment, as they can have both promotion and inhibition roles. Autophagy often plays a suppressive role during the initial stages of carcinogenicity, while in the later stages of cancer development, it can play a promoting role 
. It is extremely important to determine the regulation of this duality of autophagy in the development of CRC and to identify the molecules involved, the signals and the mechanisms that allow autophagy to play a pro-malignant dominant role in some conditions and the opposite role in others. The most interesting research on autophagy in colorectal carcinogenesis has been focused on several molecules, mainly microtubule-associated protein 1A/1B-light chain 3 (LC3), sequestosome 1 (SQSTM1/p62) and nuclear factor-κB (NF-κB), and these studies have produced conflicting results.
2. Apoptosis and Autophagy in Adenoma–Carcinoma Sequence
The crypts of the human colorectal mucosa exhibit a polarized structural organization. The reserve pool of the stem cells of the regenerative epithelium is very small and located at the base of the crypt. Stem cells move from the bottom of the crypt up, and on the apical margin of the mucosa about 10 million cells per day die from apoptosis, after which they fall into the lumen 
. For this reason, adequate cell death is required to maintain the homeostasis of normal colorectal mucosa. However, defective signaling or unbalanced regulation of apoptosis is probably one of the causes of the onset and progression of an adenoma to the carcinoma sequence that results in CRC. It should be noted that the proteins most involved in apoptosis, such as Bcl-2 antagonist killer 1 (Bak) or Bcl-2, are not equally expressed in all anatomical regions of the colorectal mucosa, confirming the fact that there is a diversified regulation of death in the bowel 
. Moreover, not only apoptosis, as a classical form of programmed cell death, has been shown to be operative and consistent in the colorectal glands, but also autophagy, a controlled process of cell self-digestion of great importance in situations of cellular stress, lack of nutrients, or energy deprivation (Figure 1
Figure 1. Different morphological features of human colon cancer cells during apoptosis and autophagy considered as prototypic cell death processes, in particular, morphogenetic changes occurring in the nucleus during apoptosis, that is, fragmentation process or, conversely, vacuole formation during autophagy.
In healthy differentiated adult cells, autophagy delivers to the lysosome damaged cytosolic components and organelles, preventing ageing, and actively participates in intracellular quality control and renovation in order to avoid the formation of protein aggregates that could cause several diseases, including neurodegenerative ones 
. In contrast to apoptosis, the intensity of autophagy cell expression decreases in the crypt from the bottom to the top. Autophagy is therefore confined to the lower part of the crypt 
The tumor suppressor p53 function is frequently impaired in human cancers, and it is thought that p53 mutations play a pivotal role in the adenoma–carcinoma transition 
and occur in 60% of reported CRCs 
. For this reason, the transition from adenoma to carcinoma in the colorectal region is considered a mechanism in which the regulation of the apoptotic process, given its relationship to the p53 mutations, is essential 
. As regards the autophagic process, its involvement in the initiation of the adenoma–carcinoma transition is still highly controversial. Studies of the relationship between autophagy and colorectal carcinogenesis have produced contradictory results: the conditional deletion of autophagy-related gene-7 (ATG7) in the intestinal epithelium does not increase the number of intestinal polyps 
, while the heterozygous deletion of autophagy-related gene-5 (ATG5)—an essential ATG protein in autophagy—in adenomatous polyposis coli (Apc) Min/+mice increases the number and size of adenomas through the activation of the epidermal growth factor receptor (EGFR)/extracellular signal-regulated kinase 1/2 (ERK1/2) and wingless and INT-1 (Wnt)/β-catenin pathways 
3. Regulatory Role of p53 in Apoptotic and Autophagic Processes
Over the years, the interest of researchers in determining different types of cell death has never diminished, since it remains an essential physiological process required to maintain tissue homeostasis. The recent recommendations of the Nomenclature Committee on Cell Death define autophagy-dependent cell death (ADCD) as regulated cell death that results from the autophagy machinery (i.e., pharmacological or genetic manipulations of autophagy genes block cell death), without involving alternative death pathways 
. However, research attempts have not yet produced an accurate picture of cell death phenomena. The fate of a cell, whether it lives or dies, is crucial and can be the determining factor in the progression of tumors 
. Incorrect regulation of the cell death signaling cascades is considered fundamental for the initiation and progression of CRC. Many in vitro studies have elucidated that the interplay between apoptosis and autophagy is highly complex and their influence in cancer can be modified by several factors, such as the types of tissue and cells involved, tumor stage and malignancy grading, type of oncogenic mutation, the entity of damage or cellular stress, intra-tumoral oxygen levels or the presence of nutrient deprivation 
. The tumor suppressor protein, p53, presents a plethora of anticancer functions 
. p53 is activated in the presence of cellular stress, including DNA damage, hypoxia, senescence and DNA repair, inducing cell cycle arrest to allow cell repair or apoptosis (mainly via the intrinsic pathway) 
. The p53 gene is mutated in a high percentage of colorectal tumors and can be found in both adenomas and malignant cells, resulting in the disruption of tumor suppressor functions 
. There is evidence that p53 mRNA expression may represent a useful survival predictor in patients with stage III CRC or rectal carcinoma 
. On the other hand, Sakanashi F in one morphological study showed that autophagy and apoptosis are overrepresented in CRC and that p53 mutation may lead to the upregulation of autophagy 
. p53 mutation may alter the ability to reduce autophagy, which implies that the p53 mutant increases autophagy, leading to more aggressive CRCs.
Another interesting paper showed that apoptotic cell death induced by N1, N11-diethylnorspermine (DENSpm) is boosted when autophagy is inhibited by 3-mase9pethyladenine (3-MA), an autophagy inhibitor, or beclin-1 siRNA through polyamine depletion or polyamine catabolic activation in colon cancer cells, regardless p53 mutation status. In conclusion, the researchers stated that p53 deficiency does not alter the response of colon cancer cells to apoptotic death induced by DENSpm in autophagy suppression conditions 
. p53 is linked to the regulation of autophagy by affecting the availability of nutrients. The loss of p53 leads to the overproduction of LC3, a standard marker for autophagosomes, and forces cells to maintain elevated levels of autophagy.
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 
. SQSTM1/p62 plays specific and indispensable roles in selective autophagy and SQSTM1/p62 protein levels have been used as an indicator of autophagic flux 
. The pivotal roles of SQSTM1/p62 in ingested protein catabolism through autophagy and lysosomal targeting have been well elucidated in the literature 
. 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 
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 
. 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 
. 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 
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 
. 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) 
. 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 
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 .
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 
. 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 
. 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 
. On the other hand, ROS can trigger DNA damage, initiating the apoptotic process pathway in colon cancer cells 
. 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 2
Figure 2. 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 
. 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 
. 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) 
, 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 
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 
. 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) 
. 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 
. 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 
. 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 
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.