Microorganism-regulated autophagy in gastrointestinal cancer

Autophagy classification

Autophagy can be classified into macroautophagy, microautophagy and chaperone-mediated autophagy (CMA) based on the pathway by which cellular contents are transported into the lysosome (Fig. 1) (Jacob et al., 2017). During the process of macroautophagy, cytoplasmic contents or organelles to be degraded are wrapped by double-membraned autophagosomes from the endoplasmic reticulum (ER) (Axe et al., 2008) and Golgi apparatus (Yen et al., 2010) which will fuse with lysosomes to form autolysosomes.

The contents will be degraded into small biomolecules and released into the cytoplasm, even outside the cell for recycling (Mizushima et al., 2001). Microautophagy is the process by which the lysosomal membrane invaginates and directly wraps the damaged organelles, then transports the cargos to the lysosomes (Gorrell et al., 2021). Finally, CMA is a selective process in which unfolded proteins recognize and bind to the molecular chaperone and enter the lumen of the lysosomes directly for degradation (Kaushik, Kiffin & Cuervo, 2007). Macroautophagy, usually referred to simply as autophagy, is the subject of this review.

The molecular mechanisms of autophagy

The whole process of autophagy includes six key steps: initiation, nucleation, prolongation, maturation, fusion, and degradation (Levy, Towers & Thorburn, 2017). The following signal molecules are involved in autophagy: the ULK1 complex (ULK1-Atg13-FIP200), the type III phosphatidylinositol 3-kinase (PI3K) complex (Class III PI3K (VPS34)-Beclin-1-Atg14), the Atg12-Atg5-Atg16 ubiquitin complex and LC3-II-PE ubiquitin complex, etc.

The ULK1 complex is involved in autophagy induction (Hosokawa et al., 2009). mTOR phosphorylates Atg13, resulting in a low ULK1 activity in normal situation. When cells are starved or hypoxic, the mTOR activity is inhibited, leading to dephosphorylation of Atg13, which will activate ULK1 complex. The activated ULK1 complex will be further transferred from the cavity to the endoplasmic reticulum to induce the membrane formation of autophagosomes (Pattingre et al., 2008). AMPK is a positive regulator of autophagy, which can directly inhibit mTOR activity and induce autophagy (Ge et al., 2021). In addition, AMPK can directly bind to ULK1 complex and phosphorylate it, thus promoting the progression of autophagy membrane (Kim et al., 2011).

The Type III PI3K-Beclin-1-Atg14 complex is involved in the nucleation of autophagosomes (Kang et al., 2011). After being activated by the ULK1 complex, it locates to the ER and produces PI3P, mediating the formation of autophagy vesicles (Levine, Mizushima & Virgin, 2011). Beclin1 is a key factor in the formation of autophagy, which can bind to anti-apoptosis-related proteins such as Bcl-2, thus playing an important role in regulating autophagy and apoptosis (Kang et al., 2011).

The Atg12-Atg5-Atg16 ubiquitin complex is involved in the prolongation of autophagosomes. The formation of the complex requires the participation of ubiquitin activating enzymes E1 and E2. Atg12 is first activated by Atg7 (E1-like enzyme), and then transported to Atg5 through Atg10 (E2-like enzyme) to form a multi-body complex with Atg16, which participates in the extension of autophagosomes (Tanida, 2011; Suzuki et al., 2005).

LC3II-PE ubiquitin complex plays an essential role in the early stage of autophagy. LC3 can be cut into soluble LC3I by Atg4, and then combined with phosphatidylethanolamine (PE) under the action of Atg7 and Atg3 to form LC3II-PE, which participates in the prolongation of autophagosomes (Kabeya et al., 2000). LC3II distributes symmetrically on the inner and outer membrane of autophagosomes. When autophagosomes fuse with lysosomes, LC3II is degraded by hydrolases in lysosomes. The content of LC3II or the ratio of LC3II to LC3I could be an indicator of autophagy (Kimura et al., 2009). Moreover, LC3II-PE can transfer the ubiquitinated products to be degraded into the autophagolysosomes through the junction of p62 (Matsumoto et al., 2011). P62 degradation is another important indicator of autophagy (Mizushima, Yoshimori & Levine, 2010).

And in the process of fusion and degradation, autophagosomes act mainly by forming the autophagolysosomal system with lysosomes (Kumar et al., 2021). In the autophagolysosomal system, Transcriptional factor EB (TFEB) plays an important role in the regulation of the expression of multiple genes, including autophagolysosomal components. The nuclear localization of TFEB is regulated by the phosphorylation of extracellular signal-regulated kinase 2 (ERK2), and its activity is modulated by the levels of extracellular nutrients. Interestingly, reactive oxygen species (ROS) play key roles in the autophagolysosomal system and may be critical for synergistic therapeutic interventions (Kumar et al., 2021; Settembre et al., 2011).

Autophagy as a tumor promoter

Autophagy is thought to function as a promoter of tumor progression and is associated with drug resistance in several types of cancer (Maes et al., 2013). However, some chemotherapeutic drugs can induce protective autophagy, thereby antagonizing drug-induced apoptosis in cancer cells (Table 1). A recent study has shown that oxaliplatin-induced protective autophagy could partially antagonize apoptosis in gastric cancer MGC803 cells, promoting tumor progression (Xu et al., 2011). The expression levels of LC3II have been reported to be positively correlated with the clinical stages in oral squamous carcinoma (OSCC) (de Lima et al., 2017). Some normal cells contribute to tumor cell growth by generating nutritional autophagy at the early stage of tumor development (Maes et al., 2013; Katheder et al., 2017). ATG16L1, an essential signal molecule for autophagy, is expressed in malignant oral cancer cells but not in normal cells, suggesting elevated levels of autophagy in tumors (Nomura et al., 2009). Thus, autophagy is likely to be a protective factor for tumor cells, allowing them to survive under stress. A recent study has demonstrated that knockdown of FIP200, a protein involved in autophagy initiation, prevented breast cancer progression, suggesting a role for autophagy in tumorigenesis (Wei et al., 2011).

Autophagy as a tumor suppressor

Autophagy can protect cells from cancerization by degrading dysfunctional proteins and organelles and preventing the toxic accumulation (Liang & Jung, 2010). Abnormal expression of autophagy-associated genes may lead to pathological changes (Maiuri et al., 2009; Tsuchihara, Fujii & Esumi, 2009). Some autophagy-associated genes are frequently mutated or absent in many human cancers (Ionov et al., 2004; Aita et al., 1999). The down-regulation of beclin1 expression has been observed in human breast, ovarian and prostate cancers (Aita et al., 1999). Similarly, knockdown of the atg5 gene and/or the beclin1 gene in normal cell line (Karantza-Wadsworth et al., 2007) has been shown to induce cell transformation.

Some drugs can induce autophagic death in cancer cells (Table 1). A study has showed that berberine, an alkaloid isolated from the Chinese herbal medicine Coptis chinensis, may induce autophagy through inhibition of MAPK/mTOR/p70S6K and Akt signaling pathways, thereby suppressing the growth of human gastric cancer cells in vivo and in vitro (Zhang et al., 2020). Berberine may also induce apoptosis in human malignant melanoma cells through activation of the ER stress-mediated autophagy (Fang et al., 2021). Berberine may also induce autophagic death in acute lymphoblastic leukemia through inactivation of AKT/mTORC1 signaling (Liu et al., 2020). In addition, berberine may induce autophagy in glioblastoma through the ERK1/2 signaling pathway, thereby increasing sensitivity to chemotherapeutic drugs (Qu et al., 2020). A study has showed that artesunate, a kind of antimalarial drug, can act as an autophagy inducer to suppress colorectal cancer progression in a ROS-dependent manner (Huang et al., 2022). Aloe gel glucomannan can induce colon cancer cell death via the PINK1/Parkin mitochondrial autophagy pathway (Zhang et al., 2022). Moreover, inappropriate degradation of components during the autophagy process may bring cytotoxicity, ultimately leading to autophagic cell death (Qian & Yang, 2016). In conclusion, autophagy is a double-edged sword for tumor cells.

The intestinal microorganisms and colorectal cancer

In recent years, the association between microorganisms and cancer development has been studied. Approximately 100 trillion bacteria colonize the human intestine (Yu et al., 2015). Intestinal microbes interact with the human body in long-term coevolution, which are closely associated with some physiological and pathological activities, such as obesity, diabetes, cardiovascular disease, etc. (Neish, 2009; Schulz et al., 2014; Wang et al., 2021b; De Vadder et al., 2014; Poli, 2020). Accumulating evidence has shown that imbalance of intestinal microbiota is closely related to tumorigenesis, as shown in Fig. 2 (Neish, 2009; Sonnenberg & Artis, 2012). Targeting the intestinal microorganisms may be a potent strategy in cancer treatment (Fong, Li & Yu, 2020; Song et al., 2020; Ji et al., 2020; Kaźmierczak-Siedlecka et al., 2020; Kim et al., 2020; Inamura, 2021; Baffy, 2020; Cueva et al., 2020; Peterson, Bradley & Ronai, 2020; Tao et al., 2020).

There is growing evidence of a direct link between intestinal microbiota imbalance and colorectal cancer (CRC) (Liu et al., 2021; Raskov, Burcharth & Pommergaard, 2017). It has been shown that the abundance of F. nucleatum positively correlated with CRC in clinicopathological stages (Castellarin et al., 2012; Kostic et al., 2012). The mucosal microbiota in normal tissues, adenomatous polyps, and adenocarcinoma tissues were compared, and the results showed that CRC tissues at early stage had a significant increase in the abundance of Fusobacterium, Parvimonas, Gemella, and Leptotrichia, and a decrease in the abundance of Bacteroides, Blautia, and F. prausnitzii, indicating an oncogenic role of microbiota imbalance (Nakatsu et al., 2015). In addition, the abundance of Peptostreptococcus, Parvimonas and Fusobacterium in CRC tissues was significantly different from that in paracancerous mucosa tissues (Nakatsu et al., 2015; Warren et al., 2013). It has been shown that F. nucleatum may invade tumor cells by binding Fap2 to Gal/Gal NAc expressed by tumor cells, mediating multidrug resistance of tumor cells (Abed et al., 2016). F. nucleatum can promote the metastasis of tumor cells by inducing the secretion of IL-8 and CXCL1 (Casasanta et al., 2020). Moreover, Exosomes derived from F. nucleatum-infected CRC cells may facilitate non-infected tumor cell metastasis by selectively carrying miR-1246/92b-3p/27a-3p and CXCL16 (Guo et al., 2020). Recent studies have confirmed that the anaerobic bacterium peptostreptococcus anaerobius can also promote the development of CRC (Tsoi et al., 2017). Enterotoxigenic Bacteroides fragilis can promote malignant behaviors by inhibiting miR-149-3PF packaged in vitro (Cao et al., 2021).

The gastric microorganisms and gastric cancer

As the third leading cause of cancer-related deaths worldwide, gastric cancer and its risk factors and prevention have been extensively studied (Noto & Peek, 2017). Gastric bacterial communities have been shown to be associated with gastric malignancy. H. pylori, a Gram-negative bacterium that colonizes the gastric epithelium, which is classified as a class I carcinogen by the World Health Organization (Moodley et al., 2012; De Meyer et al., 2015). H. pylori infection is thought to be the main cause of gastric cancer, but its exact mechanisms have not been fully understood (Fig. 3). H. pylori releases various virulence factors, such as vacuolating cytotoxin A (VacA) and the effector protein cytotoxin-associated gene A (CagA), to promote the development of gastric cancer (Ferreira, Machado & Figueiredo, 2014). Prolonged infection with H. pylori can lead to gastric atrophy, resulting in hyperacidity or decreased gastric acid production. Notably, H. pylori infection and the following change in the acidity of the gastric environment may further lead to alterations in the gastric microbiota (Espinoza et al., 2018). In addition, H. pylori infection was reported to increase the expression of VCAM1 in cancer-associated fibroblasts (CAFs) via JAK/STAT1 signaling pathway in gastric carcinoma, and the level of VCAM1 in patients with gastric cancer was positively correlated with tumor progression and a poor prognosis. Moreover, the interaction between CAF-derived VCAM1 and integrin αvβ1/5 could promote gastric cancer cell invasion both in vitro and in vivo (Shen et al., 2020). A recent study showed that Firmicutes and Actinobacteria were over-distributed in gastric cancer compared to chronic gastritis (Ferreira et al., 2018). These findings suggest that microbial imbalance increases the risk of gastric cancer. Till now, the functional role of microbial communities in gastric tumorigenesis and its pathogenic mechanisms have not been well understood.