s2fieldsofstudy: Biology Medicine

text:
Microbiome dysbiosis and alcoholic liver disease☆

Microbiome dysbiosis is strongly associated with alcoholic liver disease (ALD). Recent studies on comprehensive analyses of microbiome compositional and functional changes have begun to uncover the mechanistic relation between microbiome and the pathogenesis of ALD. Importantly, targeting the microbiome has become a potential strategy for the prevention and treatment of ALD. In this review, we summarize the clinical evidence of microbiome dysbiosis in ALD patients, and experimental advances in microbiome and metabolomic functional changes in animals with different species and genetic backgrounds in ALD. We also summarize the studies in humanized intestinal microbiome and fecal microbiota transplantation in mice. We introduce new developments in the studies on the role of the circulating bacterial microbiome, oral bacterial microbiome and fungal microbiome in the development of ALD. We highlight the potential mechanisms by which microbiome dysbiosis contributes to ALD, including short chain fatty acid changes, bile acid metabolism, intestinal barrier function, release of bacterial and fungal products, and inflammation. In addition, we summarize the recent developments targeting the microbiome in prevention and treatment of ALD, including dietary nutrient interference, herbal medicine, antibiotics, anti-fungal agents, probiotics, engineered bacterial therapy, fecal transplantation and oral hygiene. Although recent preclinical studies have advanced our understanding of the microbiome and ALD, clinical studies, especially prospective studies with large samples, are needed to better understand the cause-effect of microbiome dysbiosis in ALD. Identifying new precision-based strategies targeting the microbiome are expected to be developed as more effective therapies in ALD.

Introduction
Bacteria, fungi and other microorganisms create the human microbiome, a community of various microbial communities located in different areas of the human body including intestine, mouth, vagina and skin. 1 The bacterial microbiome is the most studied of these regarding its functions in the mammalian host. There are over 3 million bacterial genes, approximately ten times more than human host genes. The gut bacterial microbiome consists of more than ten phyla, and 90% of the bacteria belong to two phyla, Bacteroidetes and Firmicutes, and most of the rest belong to four phyla Actinobacteria, Fusobacteria, Proteobacteria and Verrucomicrobia. The human gut microbiome is created in utero and becomes relatively stable in early childhood. The microbiome composition and function affect the host by enhancing food metabolism, 2 supporting the barrier for defense against dangerous pathogens and providing the host with essential metabolites. 3,4 Recent studies have demonstrated that an imbalanced microbiome (dysbiosis) is associated with many diseases and disorders such as cancer, 5-8 cardiovascular diseases, [9][10][11] liver diseases, [12][13][14][15][16][17][18][19][20] and other metabolic disorders. [21][22][23] Investigation of the relation between the microbiome and these diseases and disorders provides opportunities for unique treatment strategies. The studies on intestinal, skin, oral and vaginal microbiome have begun to support the development of unique approaches to potentially modulate microbiome-related diseases.
Alcoholic liver disease (ALD) encompasses a broad spectrum of stages including fatty liver (steatosis), steatohepatitis, fibrosis, cirrhosis, and even liver cancer. 24 Although approximately 95% of heavy drinkers develop hepatic steatosis, only a small portion progresses to advanced liver disease, indicating that factors other than the amount of alcohol consumed may contribute to ALD development/progression. Despite extensive research, the cellular and molecular mechanisms underlying the development/progression of ALD are not fully understood.
Accumulating evidence demonstrated that microbiome dysbiosis is a risk factor in ALD development/progression. The majority of studies on relation between the microbiome and ALD have been focused on intestinal bacterial microbiome dysbiosis. Recent studies also indicate that oral bacterial dysbiosis and intestinal fungal dysbiosis are risk factors for ALD. The aim of this review is to summarize recent advances in ALD studies related to the microbiome, including the intestinal microbiome, oral microbiome, circulating microbiome, intestinal fungal microbiome, potential mechanisms of toxicity, and microbiome targeted treatment strategies. diversity. Alcohol metabolism-generated aldehydes in the intestine produce reactive oxygen species that trigger pro-inflammatory responses and cause intestinal epithelial barrier dysfunction. This results in increased bacterial transcytosis and translocation of bacterial products, including endotoxin (lipopolysaccharide, LPS), bacterial deoxyribonucleic acid (DNA), and other pathogen-associated molecular patterns (PAMPs), from the gut lumen to the liver, causing liver injury. Alcohol consumption-induced leaky gut has been demonstrated in human subjects with ALD, in which higher levels of serum bacterial products were found compared to healthy controls. 26,27

Gut bacterial dysbiosis and functional changes in ALD patients
Gut bacterial dysbiosis has long been observed in human alcoholic subjects. That alcohol abuse causes small intestinal bacterial overgrowth was documented over three decades ago. 28 Bacterial cultures of samples from jejunal juice displayed significantly higher bacterial counts in alcoholics compared to healthy controls. The small intestinal bacterial overgrowth may contribute to gut leakiness in patients with chronic alcohol abuse. Similar results were found by the same group of investigators using a breath test showing a higher prevalence of small intestinal bacterial overgrowth in chronic alcoholics compared to controls. 29 This observation was later confirmed in alcoholic patients with hepatic cirrhosis by other groups. [30][31][32] Development of sequencing techniques greatly advanced gut bacterial research. Alcohol consumption, either excessive or moderate, caused small intestinal bacterial overgrowth. 33 In a study involving 244 alcoholic cirrhotic patients, investigators found that intestinal dysbiosis was more severe in decompensated cirrhotics compared to compensated cirrhotics. 34 A comparative metagenomic study of 99 patients with alcohol dependence syndrome with or without liver cirrhosis found that alcoholic dependence and liver cirrhosis were associated with profound shifts in gut bacterial community structures and metabolic potential across the patients, suggesting a strong negative influence of alcohol dependence and associated liver dysfunction on gut microbiota. 35 Colon biopsy sample analysis revealed that colonic mucosa-associated bacteria were persistently altered in a subset of alcoholics, and this was correlated with serum LPS levels. 36 This clinical finding was also documented in the authors' preclinical observations in rats. 37 Although studies on bacterial overgrowth and dysbiosis are increasing, as yet there is no specific intestinal bacterial pattern identified to have an etiological role in the development of ALD. However, the fact that alcohol consumption causes intestinal bacterial dysbiosis provides an opportunity for the treatment and/or prevention of ALD by targeting intestinal microbiota.
Alcohol-dependent subjects who had gut leakiness on admission were more likely to exhibit psychological symptoms following three weeks of abstinence compared to those without gut leakiness. Moreover, subjects with initial altered gut permeability had greater alteration in gut microbiome and fecal metabolites. 38 The functional changes in microbiota (gut bacteriamediated metabolite changes) have also been studied in ALD patients. Alcohol-dependent subjects with high intestinal permeability showed higher phenol and lower 4-methyl-phenol concentrations in feces than the subjects with low intestinal permeability, while indole and 3-methyl-indole concentrations were lower in subjects with high gut permeability. Volatile organic compounds (VOC) were analyzed in fecal samples collected from alcoholics and non-alcoholic healthy controls. Several notable metabolite alterations were found in the alcoholics that were different from healthy controls. While propionate and isobutyrate (beneficial short chain fatty acids (SCFAs)), caryophyllene (alcohol consumption natural suppressant) and camphene (hepatic steatosis attenuator) were decreased, tetradecane (oxidative stress biomarker) was elevated in alcoholics. 39 These metabolite alterations could be associated with liver injury from alcohol abuse.

Gut bacterial dysbiosis and functional changes in animal models of ALD
2.2.1. Rodent models-Studies in experimental animal models of ALD provide more detailed mechanistic information on gut dysbiosis compared to clinical observations. Yan et al. 40 showed that 3 weeks of intragastric alcohol ingestion led to bacterial dysbiosis in mice, which was associated with the reduction of antimicrobial peptides, regenerating islet-derived 3 (Reg3) beta (Reg3B) and gamma (Reg3G). Alcohol-feeding increased Bacteroidetes and Verrucomicrobia and decreased Firmicutes levels compared to pair-feeding. Interestingly, an overgrowth of Akkermansia muciniphila was observed in one mouse model of ALD, and this bacterium is believed to be responsible for mucin degradation. Moreover, the population of Lactobacilli was depleted in alcohol-fed mice, which is generally considered as a beneficial bacterial group.
In another study using the Lieber DeCarli alcohol feeding mouse model, 41 metagenomic analysis demonstrated a decline in the abundance of both Bacteroidetes and Firmicutes phyla, with a proportional increase in the Gram-negative Proteobacteria and Gram-positive Actinobacteria phyla. Genera analysis showed the greatest expansion in Gram-negative alkaline tolerant Alcaligenes and Gram-positive Corynebacterium. These alterations were accompanied by the increases in colonic pH and levels of liver steatosis. 41 Chronic ethanol consumption leads to alterations in fecal bacterial microbiome, as well as gastrointestinal content of metabolites. One of the major functions of gut bacteria is to metabolize food to produce metabolites that are beneficial (or harmful) to the host. A metabolomic study revealed profound alterations in metabolite contents within the gastrointestinal tract in rats following four or eight weeks of ethanol exposure. 42 Major metabolic pathways that are critical for host physiology were affected, including markedly altered bile acids (BAs), increased levels of certain fatty acids and steroids, decreased carnitines and metabolites involved in lipid metabolism, a significant decrease of all amino acids and branched chain amino acids, and significantly decreased SCFAs except for acetic acid, which rapidly elevated as a product of ethanol metabolism. 42 Metabolomic studies by our group also revealed changes in metabolites in the liver and intestine. Levels of dietderived long chain fatty acids increased in mouse livers and decreased in mouse feces when mice were chronically exposed to alcohol. Several amino acids including branched chain amino acid L-Isoleucine were down-regulated in the liver and fecal samples from animals exposed to alcohol. 43 Interestingly, heptadecanoic acid (C17:0), a long chain fatty acid produced only by bacteria, was reduced by alcohol ingestion, indicating alcohol exposure changes gut fatty acid-metabolizing bacteria. Supplementation of saturated long-chain fatty acids maintains intestinal eubiosis and reduces alcohol-induced liver injury. 44 The effect of the types of dietary fat on gut microbiota homeostasis was evaluated in mice with ALD. Saturated fat (medium chain triglycerides enriched) feeding reduced Proteobacteria and Actinobacteria and increased Bacteroidetes in feces of mice exposed to 8-weeks of alcohol feeding. 45 A reduction in endotoxemia and liver steatosis and injury was observed in animals fed a saturated fat diet as compared to a comparable amount of unsaturated fat (mainly linoleic acid) diet.
More recent studies analyzed polar metabolites in the feces from mice fed with or without alcohol. 46 Taurine, nicotinic acid, and several major SCFAs, were significantly decreased in mice fed alcohol. Moreover, we showed that the deficiency of an antimicrobial peptide, cathelicidin, in mice (Camp −/− mice) led to similar alterations in intestinal metabolites to that observed in alcohol-fed wild type mice. 46 The translocation of bacteria and bacterial products from the intestine to the liver is a hallmark of ALD. 47 In a recent study in which mice were subjected to chronic ethanol feeding for eight weeks, researchers found that alcohol exposure changes bacterial αdiversity in the ileum and the liver and leads to compositional changes, especially in the ileum which is largely driven by an increase in the endotoxin-producing Gram-negative phyla. Among these phyla, Prevotella was not only increased in the mucus layer of the ileum but also in liver samples. 47

Mouse models with altered genetic background-Alcohol
exposureassociated gut bacterial alteration was also studied in mice with various genetic backgrounds. Mucin production from Goblet cells in the intestine is important for the integrity of intestinal mucus layer, which forms a physical barrier between the underlying epithelium and the gut lumen. Mucin 2 is the dominant mucin in the small and large intestine. Surprisingly, wild-type mice have higher alcohol-induced hepatic steatosis and injury than mucin 2 knockout mice (Muc2 −/− ). 48 Muc2 −/− mice are protected from intestinal bacterial overgrowth after alcohol feeding compared to wild-type mice. This protective effect may be due to the higher expression of antimicrobial peptides, Reg3B and Reg3G lectins, and less mucus layer thickness that allows antimicrobial peptides to readily enter into the gut lumen and enhance intraluminal killing of bacteria. Interestingly, overexpression of Reg3G in the intestinal epithelial cells restricts bacterial colonization of the mucosal surface, reduces bacterial translocation, and protects mice from alcohol-induced steatohepatitis. 49 The importance of antimicrobial peptides Reg3B and Reg3G was further demonstrated in Reg3b −/− and Reg3g −/− mice. Compared to wild-type mice, the knock-out mice had a similar change in fecal microbiota after alcohol feeding. However, Reg3b −/− and Reg3g −/− mice showed significantly higher numbers of mucus-associated bacteria in the mucus and epithelial layer of the small intestine and an enhanced bacterial translocation to the mesenteric lymph nodes and liver. 49 Alcohol appears to impair control of the mucosaassociated microbiota, and the subsequent breach of the mucosal barrier facilitates the progression of ALD.
Alcohol consumption also decreased other types of antimicrobial peptides. Cathelicidinassociated antimicrobial peptides (CRAMP) is a mouse ortholog of LL-37, the only member of the human cathelicidin family. Intestinal CRAMP is down-regulated by alcohol feeding, which was associated with gut dysbiosis and increased circulating LPS levels. 19,50 CRAMP knock-out exacerbated ALD, 51 which was associated with decreased butyrate-producing bacteria population (unpublished data) and reduced fecal butyric acid concentration. 46 The Paneth cell-produced antimicrobial peptide, α-defensin, is another important group of antimicrobial peptides. Although expression of α-defensin 5 did not differ significantly in mice fed alcohol intragastrically for 3 weeks, 40 dysfunction of α-defensin 5 exacerbated alcohol-induced luminal and mucus-associated microbiota and resulted in a severe liver injury. 52 Zinc supplementation increased α-defensin 5 production and reduced ALD.
Alcohol-associated metagenomic changes are associated with alterations in bile acid profiles with significant decreased taurine-conjugated BAs in intestine and in the serum in rat model of ALD. 42,53 Alcohol feeding increased abundance of bacteria harboring cholylglycine hydrolase (CGH), 54 a major enzyme that deconjugates bile acid. 55 Interestingly, serum conjugated BAs were markedly elevated in alcoholic cirrhosis patients. 56 The mechanism underlying the difference of conjugated bile acid in human and rodents are unknown, but the changes in gut microbiota may be a causative factor for these changes. In contrast, recent study showed increased amount of unconjugated BAs in the small intestine of mice fed alcohol for 8 weeks. 54 Increased unconjugated BAs are associated with lower farnesoid X receptor (FXR) activity in enterocytes, lower fibroblast growth factor (FGF)-15 protein secretion and increased hepatic cytochrome P450 enzyme (Cyp)-7A1 protein expression and circulating bile acid levels. Depletion of the commensal microbiota with non-absorbable antibiotics attenuated hepatic Cyp7A1 expression and reduced ALD in mice, suggesting an important role of gut bacteria in bile acid de novo synthesis. Pharmacological intervention with the intestine-restricted FXR agonist, fexaramine, to restore intestinal FXR activity protected mice from ethanol-induced liver injury. 54 While inhibition of bile acid synthesis by activating intestinal FXR is beneficial in ALD, genetic knock out of Cyp7a1 exacerbated ALD in mice subjected to binge-on-chronic alcohol feeding. 57 These contradictory results are likely due to the differences in animal species, feeding protocol and animal sex difference in these observations. Interestingly, clinical study showed an increase in serum total BAs, while a decrease in de novo bile acid synthesis in alcoholic hepatitis (AH) patients compared to healthy controls. 58

Monkey and fish models-
The relation between ALD and the gut microbiome was also explored in other animal models. The rhesus monkey provides great translational validity in preclinical studies due to its similarities to humans in genetic, physiological, metabolic and behavioral aspects. Wang et al. 59 demonstrated that rhesus monkeys fed alcohol displayed a change in the intestinal bacterial community structure at phylum, order, family, genus and species levels, along with liver steatosis. 59 Firmicutes, Proteobacteria, and Verrucomicrobia tended to increase whereas Bacteroidetes and Actinobacteria decreased. At the genera level, Lactobacillus and Streptococcus decreased in ALD monkeys compared with normal controls.
Small-sized medaka fish have been used to study the changes of intestinal microbiome by alcohol exposure. 60 Exposing the fish to alcohol for two months caused liver steatosis and injury, along with changes of fecal microbiota composition. Decreased numbers of Fusobacterium, Tenericutes and Firmicutes, and increased abundance of Proteobacteria and Bacteroides were observed. Moreover, at the species level, Alcaligenes incertae sedis and Cloacibacterium incertae sedis decreased while Cetobacterium incertae sedis and Erysipelotrichaceae incertae sedis increased with ethanol administration. These changes were similar to those observed in humans. 60

ALD models using germ-free mice and microbiota humanized mice
The germ-free rodent model provides a unique tool to study the contribution of intestinal microbiota to disease. Germ-free mice/rats can be protective in autoimmune hepatitis and liver cancer induced by chemicals, 61-63 while they can exacerbate acute liver injury and liver fibrosis. [64][65][66] The protective effects may be due to the deficient natural killer T cells in germfree mice, which have lower levels of presented bacterial glycolipid antigens compared to conventional mice, and/or due to the absence of bacterial LPS and lack of Toll-like receptor 4 (TLR4) signaling in germ-free mice. On the other hand, germ-free mice are more sensitive to acute liver injury and liver regeneration. These effects may be also due to the absence of LPS, since LPS has been shown to stimulate liver regeneration. 67 In ALD, germ-free status can be associated with worsened or improved hepatic phenotype depending on the type of animal and the experimental models. Feeding germ-free Swiss mice alcohol for 7 days resulted in a reduced liver injury compared to conventional mice. 68 This was associated with a reduced intestinal permeability and lower neutrophil accumulation. On the other hand, C57BL/6 germ-free mice showed significantly more pronounced liver injury, inflammation and steatosis than conventional mice treated with acute alcohol administration. 69 The increased susceptibility to acute alcohol-induced liver injury in germ-free mice may be related to a more efficient xenobiotic metabolism, elevated baseline levels of inflammatory factors and up-regulated genes involved in lipid synthesis. The discrepancy between these two studies could be due to the different animal strains and germ-free status maintenance. Studies using different rodent strains in ALD have shown that the strain can have a significant effect on multiple aspects of ALD pathogenesis, including alcohol metabolism, inflammation, fat accumulation and stress response. It is also very difficult to sterilize the liquid Lieber DeCarli diet every day and to keep germ-free status in animal facilities.
To overcome the limitations of germ-free mice, microbiota humanized mice have been used in studies of ALD. 70 Humanization of mice using human intestinal microbiota transplants from ALD patients showed significant differences between germ-free and conventional mice. 18 The germ-free mice receiving the intestinal microbiota from a patient with severe AH developed more severe liver inflammation, greater liver necrosis, greater intestinal permeability and more translocation of bacteria to the liver than in mice transplanted with intestinal microbiota from an alcoholic patient without AH. Interestingly, in conventional mice humanized with the intestinal microbiota from a severe AH patient, a second subsequent transfer of intestinal microbiota from patients without AH improved alcoholinduced liver lesions. Although such a strategy cannot be used in the clinical setting due to the ethical concerns, humanized microbiota animal models can offer a better understanding of the associations between microbiota and ALD. The importance of intestinal microbiota in ALD is further demonstrated by fecal microbiota transplantation (FMT) between mice that are housed in different facilities. 71 Two groups of mice were housed in two nearby facilities, and the mice in one facility developed ALD (alcohol-sensitive) and the mice in other facility did not (alcohol-resistant), despite a similar alcohol intake. Alcohol induced hepatic steatosis and liver inflammation, which were associated with gut dysbiosis in the alcohol-sensitive mice. Importantly, transplantation of intestinal microbiota from the resistant mice to the sensitive mice restored gut flora homeostasis and prevented the development of ALD.

Circulating microbiota dysbiosis and ALD
Microscopic studies using bacterial culture have demonstrated that many bacteria can survive in a dormant form in blood and inside of red blood cells, and these findings were further confirmed by next-generation sequencing techniques. [72][73][74][75] Changes in blood microbiota profiles have been linked to liver fibrosis in obese patients. 76 Recent study by Puri and colleagues 77 determined the circulating blood bacterial microbiota by qualitative and quantitative analyses of bacterial DNA in human subjects with moderate AH or severe AH, and compared the results with those from heavy drinkers without liver disease and nondrinkers. Multiple changes in the circulating microbiome were found in heavy alcohol consumers with AH and in heavy drinkers without obvious liver disease, and these changes are associated with a shift in the metabolic functions of bacteria. Patients with AH had higher levels of circulating bacterial DNA compared to non-drinkers and the drinkers without liver disease. All alcohol consumers had a significantly decreased Bacteroidetes, and an enrichment of Fusobacteria. Functional metagenomics analysis revealed that alcohol consumption associated with an increase of bacteria with genes related to methanogenesis and denitrification. Heavy alcohol consumption appears to be the primary driver of changes in the circulating microbiome associated with a shift in its inferred metabolic functions. More studies are required to elucidate the role of the circulating microbiome and its related metabolites in the pathogenesis of ALD. Especially, well-designed preclinical animal studies on the circulating microbiome will further help to gain insights into the mechanisms of the changes produced by alcohol consumption and liver diseases. Nevertheless, the blood microbiome analysis provides potential biomarkers for the detection of AH and other forms of liver disease.

Oral microbiota dysbiosis and ALD
Bacterial infection is frequently observed in patients with ALD. The oral cavity is home to several million bacteria that can cause periodontitis and systemic disease. 78 The oral microbiota plays an important role in maintaining our health by forming a protective layer in the mouth, which prevents colonization by pathogens. If the microbial composition is disturbed, the oral microbiota may promote a state of disease in the oral or even extra-oral tissues such as the liver. The relation between periodontopathic bacteria and liver diseases has been explored in several studies. Oral microbiota dysbiosis is associated with autoimmune liver disease, 79 non-alcoholic fatty liver disease (NAFLD), 80 non-alcoholic steatohepatitis (NASH), 81 and liver cirrhosis. 82 The oral cavity could be an important underdefined source of inflammation in liver cirrhosis. The emerging role of the oral-gut axis in the development and progression of cirrhosis was highlighted by Bajaj et al. 83 in a recent review article. It is possible that the link between the liver and the oral cavity could be via the gut through impaired intestinal integrity that, in turn, could allow direct translocation of bacteria and/or their products and inflammatory mediators from the oral cavity to the systemic circulation.
Porphyromonas gingivalis (P. gingivalis) is a major pathogen of severe periodontal disease (PD). It functions as a keystone pathogen that not only sets the stage for the entire cascade of PD by altering the local immune microenvironment, but also enters the blood circulation and contributes to multiple systemic diseases. 84,85 P. gingivalis can also be swallowed with saliva and enter the intestine, 86,87 causing liver disease via the gut-liver axis. 88 Periodontitis is associated with increased hepatic fibrosis in human subjects with NAFLD and P. gingivalis worsens steatohepatitis in mice fed a high-fat diet. Interestingly, a program of dental hygiene therapy improved NAFLD in humans. 80,81,89 Recent studies explored the association of oral disease and oral microbiome with alcohol consumption. Alcoholic patients have a pathogenic oral microbiome and worse PD than nonalcoholic patients, 90 and alcoholic patients with a smoking history have higher odds ratio of having PD. 91 More recently, a study showed an association of P. gingivalis with acute alcoholic hepatitis (AAH) development/progression. 88 Patients with severe AAH showed significantly higher plasma levels of IgG, IgA, and IgM against two P. gingivalis strains (W83 and 33277) compared to healthy controls. Patients with moderate AAH also had significantly elevated anti-P. gingivalis IgA concentrations of both strains compared to healthy controls. Male patients with moderate AAH showed a significant inverse association in lifetime drinking history (LTDH) and anti-P. gingivalis IgM. The aspartate aminotransferase (AST): alanine aminotransferase (ALT) ratio was positively associated with IgM of both P. gingivalis strains in male patients with moderate AAH. Female patients with severe AAH showed a significant association between MELD scores and W83 IgM. P. gingivalis may be associated with ALD and may function as a confounding factor in AAH. This study supports the concept that infection with oral P. gingivalis is associated with both progression and severity of AAH, and this association was modestly impacted by sex. Further studies are indicated to determine whether treatment of PD may help to prevent or attenuate ALD in humans and to understand the cause-effect relation between oral P. gingivalis and ALD.

Fungal dysbiosis and ALD
Although intestinal microbiome consists of bacteria, fungi and viruses, almost all the studies on ALD and the microbiome focus exclusively on gut bacteria. Dysbiosis of intestinal fungi, or mycobiota, has been associated with diseases such as colitis. [92][93][94] A recent study demonstrated that intestinal fungi contribute to the development of ALD. 95 There are significant changes in abundance and composition of intestinal fungi between alcoholdependent patients and healthy controls. Fungal diversity was lower in alcoholics than controls. A dramatic overgrowth of Candida, with concomitant decreases in Epicoccum, Galactomyces and Debaryomyces was observed in alcoholics. Interestingly, the degree of exposure to fungal products correlates with mortality of patients with cirrhosis from alcohol abuse but not viral hepatitis. Although this study supports the importance of the mycobiota for patients with ALD, a larger, prospective study is required to confirm data from this retrospective analysis of human subjects.
Chronic alcohol feeding increased fungi populations and translocation of β-glucan, a fungi cell wall component, into systemic circulation in mice. Once relocated inside the liver, βglucan binds certain immune cells and triggers inflammation, which ultimately promotes ALD. Treatment of alcohol-fed mice with an antifungal compound, amphotericin B, protects mice from alcohol-induced liver disease. 95 This study demonstrated a potential important role of fungal dysbiosis in the development of ALD. Manipulation of the intestinal mycobiome might be an effective strategy for treatment of alcohol-related liver disease.

Treatments targeting microbiome in ALD
Restoration of gut eubiosis is the major aim of gut-microbiota based therapies in ALD. Several approaches including diet modulation, probiotics, prebiotics, antibiotics, antifungals intervention, engineered-bacteria therapy, fecal bacteria transplantation and oral hygiene have proven to be effective in alleviating alcohol-induced liver injury through positively modifying gut microbiota.
Dietary supplements have received increasing interest in ALD management. It has been shown that unsaturated fat together with alcohol consumption is harmful in ALD. Supplementation of saturated fat (medium chain triglycerides enriched) in mice with chronic alcohol feeding restored gut microbiota profiles and reduced alcohol-induced gut barrier dysfunction. 45 Alcohol consumption is often associated with micronutrient deficiency, such as vitamins and zinc. Zinc deficiency occurs as early as after two weeks of alcohol feeding in mice, and alcohol-induced zinc deficiency could also be detected in the small intestine, and this exacerbates gut barrier dysfunction caused by alcohol. Supplementation with zinc restored microbiota and gut barrier function. 96,97 Many other dietary supplements have been studied for the prevention/treatment of ALD in animal models. Dietary flaxseed oil was shown to protect the liver from alcohol-induced injury, likely through gut microbiota modulation. 98 Herbal supplements have also been used to target gut microbiota in ALD. 99 It is logical that antibiotic use may restrict pathogenic bacteria in ALD management. Intestinal sterilization was used as a prevention strategy for alcohol-induced liver injury in rats as early in 1995. 100 Fecal culture of stool samples from ethanol-fed rats treated with antibiotics showed virtually no growth of Gram-negative bacteria, and rats receiving antibiotics had reduced endotoxin levels and liver injury. 100 Non-absorbable antibiotics have been used to reduce ALD in mice. 19 In patients with ALD, treatment with antibiotics led to an improvement in the Child-Pugh score and cirrhosis index. 101,102 These findings might be related to the reduction of endotoxemia resulting from intestinal decontamination, and highlighted the therapeutic potential of antibiotics in treating ALD. However, antibiotic treatment frequently induces diverse off-target side-effects by killing a wide set of microbes outside of the desired target, including increased susceptibility to pathogenic bacteria, such as Clostridium difficile. 103 Another approach targeting microbiome dysbiosis in ALD is the use of probiotics. A variety of probiotic strains have been administrated to rodents for the prevention/treatment of experimental ALD. 104 Among those, Lactobaciilus rhamnosus GG (LGG) is the most frequently used strain.
LGG administration to rodents on chronic alcohol exposure reduced liver injury and steatosis. 105 The beneficial effects of LGG were associated with gut microbiota modification, improved intestinal tight junction protein expression, and reduced serum endotoxin levels. VSL#3 was demonstrated to be effective in modulating gut microbiota and protecting against alcohol-induced intestinal barrier dysfunction. 106 A combination treatment using Lactobacillus acidophilus, Lactobacillus helveticus and Bifidobacterium in rats with alcoholic pancreatitis-related liver damage effectively protected against endotoxin/bacteria translocation, as well as liver damage in the course of acute pancreatitis and concomitant heavy alcohol consumption. 107 While many reports have studied the effects of probiotics in experimental ALD, clinical data are limited. Effectiveness of the probiotic Lactobacillus casei Shirota on the alcoholic cirrhosis patients was evaluated in a small open-labeled study (N = 12). 108 Compared to the control group, cirrhotic patients who received the probiotics for 4 weeks had a significantly lower TLR4 expression as well as soluble tumor necrosis factor receptor 1 (sTNFR1) and sTNFR2 levels, along with a restored neutrophil phagocytic activity, suggesting that the probiotic is safe and may be effective in the treatment of patients with defective immunity. Administration of a synbiotic mixture of different bacteria strains improved liver damage and function in 10 alcoholic liver cirrhosis patients. 109 Notably, the effects seemed to be partially persistent. In humans, Kirpich et al. 31 demonstrated that after 5 days of treatment with Bifidobacterium bifdum and Lactobacillus plantarum 8PA3, mild AH patients had a significant end-of-treatment reduction of ALT, AST, lactate dehydrogenase and total bilirubin. These improvements were associated with changes in the fecal commensal bacteria Bifidobacteria and Lactobacilli.
Probiotic treatments on multiple pathological disorders are not always effective due to the viability of these beneficial bacteria. 110,111 Colonization of the gut is essential for live bacteria in order to confer their beneficial effects. However, in many disease conditions including ALD, there is an augmentation of pathogenic bacteria, which could dampen the ability of probiotics to colonize the gut. Drugs, in particular antibiotics, used by patients may be harmful to live probiotics. Therefore, an unstable and variable effect of live probiotics may occur. Moreover, the clinically recommended dose of probiotics usually consists of billions of live bacteria, and ingesting such large amounts of bacteria raises safety concerns, especially for patients with compromised intestinal function and immune response. 112,113 In fact, soluble factors secreted from probiotics and dead probiotics have been shown to be effective in treating several diseases conditions such as inflammatory bowel disease, colitis, and arthritis. [114][115][116] Yan et al. 117 demonstrated that soluble proteins produced by probiotic bacteria regulate intestinal epithelial cell survival and growth. Oral administration of heatkilled Lactobacillus brevis SBC8803 has also been shown to ameliorate ALD in mice by inducing the expression of cytoprotective heat shock proteins and improving intestinal barrier function. 118 The effectiveness of LGG culture supernatant in the prevention of acute and chronic alcohol-induced hepatic steatosis and liver injury has been investigated by our group. 50,119,120 Pretreatment with LGG supernatant (LGG-s) reduced hepatic fat accumulation in mice in an acute-binge alcohol model. 50 We also demonstrated that coadministration of LGG-s with alcohol using the Lieber DeCarli liquid diet for 4 weeks significantly prevented alcohol-induced intestinal barrier dysfunction, endotoxemia, fatty liver and inflammation in mice. 119,120 The use of probiotic culture supernatant opens a new avenue for probiotic application. Further characterization of the LGG-s active components will enhance our understanding of the protective effects of probiotics in ALD and advance the development of new therapeutic strategies for ALD.
Genetically modified-bacteria have also been evaluated for the treatment of ALD. Administration of engineered Lactobacillus reuteri to produce interleukin-22 (IL-22) (L. reuteri/IL-22) in mice on chronic alcohol exposure reduced liver damage, inflammation and bacterial translocation to the liver compared with mice fed an isogenic control strain. The beneficial effects were associated with up-regulated intestinal expression of Reg3G, which plays a pivotal role in gut microbiota homeostasis. 121 FMT as a therapeutic strategy has been used in disease treatment involving intestinal bacterial infection. 122 Preclinical studies showed that transplanting feces from alcoholresistant donor mice to alcohol-sensitive recipient mice resulted in prevention of alcoholinduced gut dysbiosis and hepatic steatohepatitis. 71,123,124 There also have been small studies suggesting beneficial effects of fecal transplantation in AH and in patients with hepatic encephalopathy. 125,126 Nevertheless, this approach should be cautiously applied to the therapy of ALD patients in the future. Recent reports of one patient's death and another patient suffering an invasive infection caused by extended-spectrum β-Lactamase-producing Escherichia Coli after receiving fecal microbiota transplants from the same donor has raised an alert on FMT. The Food and Drug Administration (FDA) has therefore halted clinical trials on fecal microbial transplantation due to this occurrence.

Summary
Increasing evidence suggests that the bacterial microbiome has a major role in ALD. Alcohol-induced bacterial microbiome alterations are associated with changes of bacterial pathogen-derived molecules, intestinal immune defenses, Paneth and Goblet cell secretion, intestinal tight junction protein expression, and integrity of gut barrier. All of these factors appear to play an integrated role in the development/progression of ALD. On the other hand, liver metabolism and immune regulation in ALD seem to affect intestinal bacterial microbiome. Although we have some understanding of the interactions between the intestinal bacterial microbiome and the host, further studies to better characterize microbiome changes and the interactions between these components are needed in additional to animal models to mimic different stages of ALD and in larger cohorts of patients. Routine monitoring of the changes in the bacterial microbiome may help to develop more personalized approach targeting microbiome for the prevention/treatment of ALD in different stages. In addition, the intestinal fungal microbiome and oral bacterial microbiome seem to also play an important role in ALD. Although we are still in the early stages in understanding the role of fungal microbiome and oral bacterial microbiome in ALD, targeting these microbiome communities provides additional opportunities to develop approaches for ALD treatment (Fig. 1).

Fig. 1. Bacterial and fungal microbiome in ALD.
Alcohol consumption causes intestinal and oral microbiome (bacterial and fungal) dysbiosis and alterations in the products of these microorganisms. Alcohol-caused damages in intestinal and oral barrier dysfunction allows the translocation of bacteria and fungi and the harmful metabolites to the liver and causes liver injury. Circulating microbiome could serve as biomarker of ALD development/progression. Targeting intestinal and oral microbiome provides a potential strategy for prevention/treatment of ALD. Abbreviations: LPS, lipopolysaccharide; SCFAs, short chain fatty acids; BAs, bile acids; FMT, fecal microbiota transplantation.


extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Biology Chemistry

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Blocking ROR1 enhances the roles of erlotinib in lung adenocarcinoma cell lines

Treatment strategies involving tyrosine kinase inhibitors (TKIs) for patients with non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) mutations have advanced significantly; however, challenges still remain regarding the development of resistance. It has been reported that receptor tyrosine kinase-like orphan receptor 1 (ROR1) acts as a hepatocyte growth factor receptor (MET) and c-Src substrate, and that the extracellular domain of ROR1 is associated with EGFR to sustain EGFR-ERBB3-PI3K signaling. Our previous study reported that blocking ROR1 significantly decreased the activity of key signal molecules in the AKT/mammalian target of rapamycin (mTOR) signaling pathway, which was associated with a significant increase of apoptosis and significant decrease of proliferation of lung adenocarcinoma cells. The present study hypothesized that inhibiting ROR1 could potentially prevent erlotinib resistance in NSCLC cell lines. Investigations were performed with two erlotinib-resistant cell lines XLA-07 and NCI-H1975, and an erlotinib-acquired-resistant cell line PC-9erlo, which was developed from its parental cell line PC-9. It was identified that the inhibition of ROR1 via small interfering RNA treatment significantly improved the anti-proliferation and apoptosis-inducing roles of erlotinib in TKI-resistant tumor cells. This was in accordance with the activity of key molecules of the AKT/mTOR signaling pathway, including glycogen synthase kinase-3α/β (GSK-3α/β), phosphatase and tensin homolog (PTEN), AKT, mTOR and ribosomal protein S6 kinase β-1 (p70S6K). The current data suggest that targeting ROR1 is a potential novel treatment strategy for patients with ROR1-positive NSCLC, particularly those with acquired resistance to EGFR-TKI.

Introduction
Cancer is a significant global health problem; in 2017 it was predicted that 600,920 cancer-associated mortalities would occur in the USA and 26% of those cases would be associated with lung cancer (1). Non-small cell lung cancer (NSCLC) accounts for >80% of primary lung cancer cases (2). The identification of specific molecular targets against NSCLC has promoted a shift towards personalized treatment strategies in clinics (3,4).
Abnormal activation of the epidermal growth factor receptor (EGFR) signaling pathway has been reported in NSCLC, which leads to the activation of subsequent intracellular signaling pathways, including the phosphoinositide 3-kinase (PI3K)/AKT and mitogen-activated protein kinase 1 (MAPK) signaling pathways, which serve important roles in the proliferation, differentiation, migration and apoptosis of tumor cells (5,6). To attenuate the effects of EGFR-mediated proliferation of cancer cells, EGFR tyrosine kinase inhibitors (EGFR-TKIs) that specifically bind to the tyrosine kinase domain of EGFR and inhibit its activity have been widely administered clinically (7).
Erlotinib is a first-generation EGFR-TKI for patients with EGFR mutation-positive lung adenocarcinoma. Erlotinib elicits effective treatment responses, however, these responses are lost after a long period of time due to acquired resistance (7,8). The most common mechanism of acquired resistance is a secondary T790 mutation in EGFR termed EGFR T790M. Other mechanisms include stimulation of alternative pathways either by activation of other kinases, including hepatocyte growth factor receptor (MET) and human epidermal growth Blocking ROR1 enhances the roles of erlotinib in lung adenocarcinoma cell lines factor receptor 2, or alterations of key components in the EGFR pathway, including activation of phosphatidylinositol-4,5-bisphosphate 3-kinase or loss of phosphatase and tensin homolog (PTEN), which eliminate the requirement for EGFR-mediated tumor cell activation (9)(10)(11)(12)(13)(14). To overcome EGFR-TKI resistance in lung cancer, numerous combinatorial strategies have been reported that demonstrate effective results and provide promising strategies to prevent resistance and potentially reduce the toxicity of both agents (15)(16)(17)(18)(19)(20)(21)(22)(23)(24). The receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a type 1 transmembrane protein expressed on the plasma membrane (25). Previous studies have demonstrated that ROR1 is an oncogene that is highly expressed in numerous types of hematologic malignancy and several types of solid tumor, including lung cancer (26,27). ROR1 acts as a partner for the oncogenic tyrosine kinase MET and sustains the MET-driven transformed phenotype (28). ROR1 is also required to sustain the association between EGFR and receptor tyrosine-protein kinase erbB-3 (ERBB3), the activation of ERBB3, consequentially, making ROR1 an ideal target for therapies against EGFR-TKI resistance in lung adenocarcinoma (29).
Our previous investigation of patients with lung adenocarcinoma revealed that >60% of tumor tissues expressed ROR1, and inhibition of ROR1 significantly downregulated the proliferation of NSCLC cells and induced cell apoptosis (27). The current study analyzed the effect of ROR1 inhibition combined with erlotinib on the induction of apoptosis and the inhibition of proliferation via the AKT/mTOR signaling pathway. In summary, the present study provided a novel therapeutic strategy to increase the sensitivity of ROR1 + lung adenocarcinoma to erlotinib treatment.

Materials and methods
Cell lines and cell culture. The NSCLC cell line NCI-H1975 was kindly provided by the Stem Cell Bank, Chinese Academy of Sciences. The human lung cancer cell line XLA-07 was a gift from Professor Yong Duan (First Affiliated Hospital of Kunming Medical University, Kunming, China) (30) and the PC-9 cell line was a gift from Dr Jun Zhang (Shanghai Pulmonary Hospital, Shanghai, China) (31). The cells were cultured at 37°C in a 5% CO 2 incubator (Panasonic Healthcare,) in RPMI-1640 (HyClone; GE Healthcare Life Sciences) supplemented with 10% fetal bovine serum (Beijing Transgen Biotech Co., Ltd.) and 1% penicillin/streptomycin (HyClone; GE Healthcare Life Sciences).
Establishment of an acquired erlotinib-resistant cell line termed PC-9erlo. The acquired erlotinib-resistant cell line termed PC-9erlo was established from the parental cell line PC-9. Briefly, 2x10 6 cells were seeded in a 10 cm 2 dish and then exposed to 10 µM erlotinib (Cayman Chemical Company). Following incubation at 37°C for 48 h the cells were washed with 1X PBS and then cultured in complete medium without erlotinib. To acquire and maintain the erlotinib resistance of PC-9erlo cells, the cultured cells were collected and gradually exposed to increasing concentrations of erlotinib (0.1 µM for 2 months, 0.5 µM for 2 months, 1.25 µM for 2 months and 2.5 µM for 2 months). Following the 2 months of exposure to 2.5 µM erlotinib, the half-maximal inhibitory concentration (IC 50 ) value of erlotinib in PC-9erlo was 2.62±0.82 µM.
Silencing of human ROR1. ROR1 small interfering RNA (siRNA), termed siROR1, was obtained from Ambion (Thermo Fisher Scientific, Inc.). The sequence of siROR1 was sense 5'-GUA CUG CGA UGA AAC UUC ATT-3'. The method of silencing ROR1 with siRNA was as previously described (27) with modifications. Briefly, the cells were seeded in 96-or 6-well plates, and incubated in a CO 2 incubator for 12 or 20 h. The cells were then transfected with siROR1 or a non-targeting control siRNA (siNC; Invitrogen; Thermo Fisher Scientific, Inc.) at a concentration of 20 or 25 nM, followed by culture in serum-free medium for 6 h. Transfections were performed in Opti-MEM reduced serum medium (Thermo Fisher Scientific, Inc.) using Lipofectamine RNA iMAX (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. MTS cytotoxicity assay. Cells were seeded in 96-well plates at 4-6x10 3 per well and transfected with 20 nM siROR1 or siNC, then cultured for 48 h prior to treatment with a range of concentrations (0, 1.25, 2.5, 5, 10, 20 µM) of erlotinib (Cayman Chemical Company). Cell cytotoxicity was examined using the CellTiter 96 ® AQueous one solution reagent (Promega Corporation) with the following steps: 20 µl of the reagent was added to each well and cells were incubated in the dark at 37°C for 1 h. Cell viability was examined by measuring absorbance at 490 nm using a microplate reader. Experiments were performed in triplicate. Cell growth ratio values were calculated using the formula: 100x [A490 (sample, T)-A490 (sample, T0)]/[A490 (control, T)-A490 (control, T0)] (T, value of absorbance at 490 nm of wells with different treatment cells; T0, value of absorbance at 490 nm of wells without cells). Cell images in different treated groups were observed by inverted optical microscope (magnification x100; XD-30; Sunny Optical Technology Co., Ltd.).
Apoptosis assay. Cell apoptosis was analyzed using flow cytometry. Cells were seeded in 6-well plates at 1.2-1.8x10 5 cells/well and appropriate concentrations of erlotinib (NCI-H1975, 2.5 µM; PC-9erlo, 2.5 µM and XLA-07, 10 µM) were added to the wells 48 h after 20 nM siRNA transfection. The plates were then incubated at 37°C for 2-5 days. Cells were collected and washed, then incubated with 5 µl FITC-conjugated Annexin V and propidium iodide (BD Biosciences) in the dark for 15 min at room temperature. Cell apoptosis was measured using a FACSCalibur flow cytometer (FACSCalibur; BD Biosciences).
Bio-Plex pro assays. Multiple proteins and the AKT signaling pathway were evaluated using a Bio-Plex signaling AKT 8-plex panel (cat. no. LQ00006JK0K0RR) and a Bio-Plex pro signaling reagent kit (cat. no. 171304006M) both from Bio-Rad Laboratories, Inc., according to the manufacturer's protocol. Briefly, 24 h after transfection with 25 nM siRNA, NCI-H1975 cells were treated with 2.5 µM erlotinib at 37°C for 24 h. The cells were lysed 48 h later with RIPA buffer (Beyotime Institute of Biotechnology) supplemented with 10% phosphatase inhibitor (Roche Diagnostics) and 1% protease inhibitor (EMD Millipore) at 4°C. Enhanced BCA Protein Assay kit (Beyotime Institute of Biotechnology) was used to analyze the protein concentration of each cell lysate. Suspended beads were added to a 96-well flat bottom plate at 50 µl per well and the plate was washed with washing buffer using a Bio-Plex Pro II Wash station (Bio-Rad Laboratories, Inc.). Subsequently, 10 µg cell lysate was added to each well. Following incubation of the plate overnight at room temperature with shaking (450 RPM), biotin conjugated detecting antibody cocktail from the kit (Bio-Plex signaling AKT 8-plex panel, cat. no. LQ00006JK0K0RR; Bio-Plex pro signaling reagent kit, cat. no. 171304006M; dilution 1:20; Bio-Rad Laboratories, Inc.) and reagent Streptavidin-PE (Bio-Rad Laboratories, Inc.) were added and measurements were obtained with the Bio-Plex 200 system (Bio-Rad Laboratories, Inc.) at a wavelength of 575 nm. Results were recorded as relative fluorescence units and data were analyzed using GraphPad Prism version 6 (GraphPad Software Inc.).
Statistical analysis. Data are presented as the mean ± standard error of the mean of at least three independent experiments. Statistical significance of data was determined by analysis of variance with LSD post hoc test, using GraphPad Prism version 6 (GraphPad Software Inc.). P<0.05 was considered to indicate a statistically significant difference.

Results
Silencing ROR1 with siRNA enhances the cytotoxicity of erlotinib in erlotinib-resistant cell lines. The present study first examined ROR1 expression levels by flow cytometry following ROR1 silencing with siRNA in NCI-H1975, PC-9erlo and XLA-07 cell lines. The results indicated that the ROR1 expression level was reduced by siROR1 with all inhibition rates >75% (Fig. 1). The growth inhibitory efficacy of erlotinib following ROR1 silencing was evaluated using the MTS assay. Compared with erlotinib alone, the specific cell proliferation rates of blocking ROR1 together with erlotinib at concentrations of 1. 25 Fig. 2A and B).

Blocking ROR1 enhances the apoptosis-inducing role of erlotinib in erlotinib-resistant cell lines.
To gain further insight into the additive roles of blocking ROR1 combined with erlotinib in erlotinib-resistant cells, NCI-H1975, PC-9erlo and XLA-07 cell lines were treated with complete medium (mock group), siNC alone, siROR1 alone, erlotinib alone (Erlo), siNC plus erlotinib (Erlo+siNC) and siROR1 plus erlotinib (Erlo+siROR1). The apoptosis rates of the cells were then analyzed using flow cytometry (Fig. 3). All three erlotinib-resistant cell lines demonstrated a limited response to erlotinib alone; however significantly different apoptosis rates were revealed when treated with Erlo+siROR1 compared with that in the Erlo group (NCI-H1975, 16.5 vs. 1.51%; PC-9erlo, 28.2 vs. 2.15%; XLA-07, 30.4 vs. 18.0%). The expression level of Bcl-2 was then further analyzed as Bcl-2 is considered an important antiapoptotic protein. Using western blot analysis it was revealed that the expression level of Bcl-2 was markedly decreased in the Erlo+siROR1 group compared with that in the Erlo group ( Fig. 4C and D), which indicated that the activity of Bcl-2 was downregulated.
Silencing ROR1 with siRNA prevents erlotinib resistance via the AKT/mTOR signaling pathway in NCI-H1975 cells. To investigate the molecular mechanisms of ROR1silencing-enhanced cytotoxicity and the apoptosis-inducing roles of erlotinib, key molecules in the AKT/mTOR signaling  pathway were analyzed in the erlotinib-resistant NCI-H1975 cell line using the Bio-Plex Pro assay ( Fig. 4A and B), and since the kit did not contain an antibody detecting total protein of IRS-1, β-actin was used as control instead. It was identified that the phosphorylation levels of IRS-1, GSK-3α/β, AKT, p70S6K, PTEN and mTOR were significantly lower in the Erlo+siROR1-treated group compared with that in the erlotinib-treated group. To confirm these findings, the phosphorylation of AKT and p70S6K was further analyzed using the western blot assay (Fig. 4C and D). This revealed that the activity of AKT and p70S6K was significantly downregulated in the Erlo+siROR1-treated group compared with the Erlo treatment group alone, which was consistent with the data from the Bio-Plex assay.

Discussion
Treatment with TKIs provides significant benefits for patients with EGFR mutations, particularly for those with lung cancer. However, the majority of patients with NSCLC will acquire resistance to first-generation EGFR-TKIs, including gefitinib and erlotinib, following 9-14 months of treatment (7). There are two central mechanisms that are involved in this process: EGFR secondary mutations and alternative signaling activation (5,6). In addition, in an EGFR-independent manner, dysregulation of other receptor tyrosine kinases (RTKs) or abnormal activation of downstream compounds have compensatory functions against the inhibition of EGFR by altering the PI3K/AKT and MAPK signaling axis. Certain studies have revealed that the proline-rich region of the intracellular domain of ROR1 is directly activated by MET and the pseudokinase domain is phosphorylated by Src (26,28). Yamaguchi et al (29) also demonstrated that a cysteine-rich domain of the extracellular domain of ROR1 is associated with EGFR and sustains EGFR-ERBB3-PI3K signaling. It may be beneficial to clarify whether ROR1 silencing has an additive role with erlotinib in lung adenocarcinoma, which could provide a potential new therapeutic strategy for patients with lung cancer, and TKI insensitivity and resistance.
The present study selected an erlotinib-resistant cell line NCI-H1975, which is known to be a T790M-mutant, and another erlotinib-resistant cell line XLA-07, and an erlotinib-acquired resistant cell line PC-9erlo, which was developed from its parental cell line PC-9 and mimics the situation that occurs in clinical treatment. The current results demonstrated that ROR1 inhibition plus erlotinib have additional cytotoxic effect in ROR1 positive lung adenocarcinoma cell lines. In addition, it was identified that the expression level of Bcl-2, a key regulator of antiapoptotic signaling (33), was significantly lower in Erlo+siROR1-treated cells (Fig. 4D), which was in accordance with the apoptosis-inducing role of ROR1 inhibition combined with erlotinib.
ROR1-mediated signaling pathways in lung cancer are not fully understood. Our previous data suggested that the AKT/mTOR signaling pathway is necessary for ROR1-mediated proliferation and antiapoptosis in lung adenocarcinoma. The AKT/mTOR signaling pathway is important for regulating cell proliferation, cancer growth and longevity (6,34,35). The present study investigated the association of the AKT/mTOR signaling pathway with ROR1 silencing against erlotinib resistance in lung cancer. Compared with erlotinib alone, phosphorylation of key molecules in the AKT/mTOR signaling pathway, including insulin receptor substrate 1 (IRS-1), glycogen synthase kinase-3α/β (GSK-3α/β), PTEN, AKT, mTOR and p70S6K, was significantly lower when ROR1 was silenced in combination with   The integrated density analysis demonstrated the changes in the expression levels of p-AKT, p-p70S6K and Bcl-2, and difference in phosphorylated protein levels was analyzed using ratios between the phosphorylated/total protein. Experiments were performed 3 times (n=3). Statistical analysis was performed using analysis of variance. * P<0.05 and ** P<0.01, erlotinib treated group vs. erlotinib+siROR1 treated group. ROR1, receptor tyrosine kinase-like orphan receptor 1; si, small interfering; NC, negative control; Mock, complete medium; mTOR, mammalian target of rapamycin; IRS-1, insulin receptor substrate 1; GSK-3α/β, glycogen synthase kinase-3α/β; p, phosphorylated. erlotinib. This supports the hypothesis that inhibiting the ROR1-mediated signaling pathway could partially overcome erlotinib-resistance via upregulation of the activity of IRS-1, AKT, mTOR and p70S6K, and downregulation of the activity of GSK-3α/β and PTEN, which are negative regulators of PI3K/AKT (Fig. 5). Inhibiting ROR1 with small molecules and monoclonal antibodies, or inhibiting the key regulators involved in AKT/mTOR signaling could effectively increase the sensitivity of tumor cells to erlotinib.
The present study revealed that the protein expression level of IRS-1, which is involved in cell proliferation, was also significantly reduced through the inhibition of ROR1 in combination with erlotinib. The underlying association between ROR1 and IRS-1 is unclear; however, targeting IRS-1 in NSCLC has been reported to exhibit an antitumor effect in a number of studies (36)(37)(38). It remains to be determined whether interactions between ROR1 and IRS-1 directly activate IRS-1 following binding to its ligand, or whether an indirect stabilization occurs through the association of IRS-1 with insulin-like growth factor-1 receptors, thus transmitting signals to the intracellular AKT/mTOR pathway.
In conclusion, the present study identified that ROR1 is a potential target for preventing erlotinib resistance in lung adenocarcinomas via the AKT/mTOR signaling pathway. Targeting ROR1 with small molecules or immunological procedures may increase the sensitizing of tumor cells, particularly erlotinib-resistant cells, to erlotinib. Further studies should investigate the functions of ROR1 in lung adenocarcinoma to promote the development of ROR1-targeting therapies in the future.


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N6-Methyladenosine: A Potential Breakthrough for Human Cancer

Among more than 100 types of identified RNA modification, N6-methyladenosine (m6A) modification is the predominant mRNA modification, which regulates RNA splicing, translocation, stability, and translation. m6A modification plays critical roles in the growth, differentiation, and metabolism of cells. As a dynamic and reversible modification, m6A is catalyzed by “writers” (RNA methyltransferases), removed by “erasers” (demethylases), and interacts with “readers” (m6A-binding proteins). With more advanced technology applied to research, the molecular mechanisms of RNA methyltransferase, demethylase, and m6A-binding protein have been revealed. An increasing number of studies have implicated the correlation between m6A modification and human cancers. In this review, we summarize that the occurrence and development of various human cancers are associated with aberrant m6A modification. We also discuss the progress in research related to m6A modification, providing novel therapeutic insight and potential breakthrough in anticancer therapy.

Among more than 100 types of identified RNA modification, N 6 -methyladenosine (m 6 A) modification is the predominant mRNA modification, which regulates RNA splicing, translocation, stability, and translation. m 6 A modification plays critical roles in the growth, differentiation, and metabolism of cells. As a dynamic and reversible modification, m 6 A is catalyzed by "writers" (RNA methyltransferases), removed by "erasers" (demethylases), and interacts with "readers" (m 6 A-binding proteins). With more advanced technology applied to research, the molecular mechanisms of RNA methyltransferase, demethylase, and m 6 A-binding protein have been revealed. An increasing number of studies have implicated the correlation between m 6 A modification and human cancers. In this review, we summarize that the occurrence and development of various human cancers are associated with aberrant m 6 A modification. We also discuss the progress in research related to m 6 A modification, providing novel therapeutic insight and potential breakthrough in anticancer therapy. BACKGROUND N6-adenosine is the most prevalent epigenetic modification in RNA, originally identified in mRNAs in the 1970s. 1 Similar to the methylation of DNA, N 6 -methyladenosine (m 6 A) methylation regulates post-transcriptional expression without changing the base sequence. m 6 A is the most abundant internal RNA modification, enriched at the RRACH motif in 3 0 UTRs, 5 0 UTRs, and near stop codons. [2][3][4][5][6] It modulates RNA processing and metabolism, including alternative splicing, transport, translation, and degradation. 7 As a dynamic and reversible modification, m 6 A deposition is regulated by methylases and demethylases. m 6 A-binding proteins subsequently recognize and bind the m 6 A-rich domain, inducing decay or accelerating translation efficiency. With the advancement of methods for detecting m 6 A and proteins, many enzymes have been identified and the functions and mechanisms of them have gradually emerged. Some studies looking into the mechanism of alternative splicing regulation have uncovered the function of m 6 A. m 6 A near splice sites in nascent pre-mRNA mediates heterogeneous nuclear ribonucleoprotein G (hnRNPG) binding. hnRNPG interacts with RNA polymerase II (RNAPII) with Arg-Gly-Gly (RGG) motifs, thereby modulating RNAPII occupancy and alternative splicing. 8 Moreover, the location of m 6 A on nascent RNA is likely to modulate splicing kinetics. m 6 A co-transcriptionally depositing near splice junctions promotes fast splicing, while m 6 A in introns indicates long, slowly processed introns and alternative splicing events. 9 m 6 A was also found in precursor mRNA (pre-mRNA), tRNA, microRNAs (miRNAs), and long noncoding RNAs (lncRNAs) later, but the functions of m 6 A in circular RNA (circRNA) remain elusive. [10][11][12][13][14] . m 6 A in circRNAs are frequently derived from exons that are not methylated in mRNAs, and a single m 6 A modification is enough to initiate circRNA translation, although the "writers" and "readers" are the same as those that participate in m 6 A modification in mRNA. 15,16 With specific m 6 A-related mechanism, circRNA is involved in tumorigenesis, which indicates the important role of circRNA in human cancer. 17 With burgeoning medical technology, substantial progress has been made in the detection and diagnosis of cancer. Accumulating studies indicate that aberrant m 6 A levels closely correlate with carcinogenesis and the progression and metastasis of cancer cells. The dysregulation of writers, "erasers," and readers is proved to be the culprit, activating oncogenes or inhibiting tumor suppressor genes by activating signaling pathways. 18,19 Potential therapeutic targets have also been offered by researching the mechanisms of carcinogenesis. In this review, we discuss the relationships between human cancers and new discoveries in the regulation of m 6 A modification.
(MAC). 20 The interface of the heterodimer is formed by strands b4-5 and helix a4 of each methyltransferase domain (MTD), and it contains hydrogen bonds as well as hydrophobic interactions. The hydrophobic region centering on strands b4-5 is protected from solvent exposure by an N-terminal extension of METTL14. In addition, METTL3 and METTL14 each forms a partially disordered loop that can insert an aromatic residue into a hydrophobic pocket in the other subunit. All of these unique structural features enhance the stability and compactness of MAC. 14,21 METTL3 is the only catalytic subunit of MAC, while METTL14 maintains MAC integrity and is likely to mediate RNA binding. Sequence analysis indicated that the METTL3 catalytic site contains a more conserved DPPW motif, whereas the catalytic motif of METTL14 is a divergent EPPL sequence. Moreover, two Cys-Cys-Cys-His (CCCH)-type zinc fingers of METTL3, adjacent to MTD3 and connected by an anti-parallel b sheet, are also necessary for methylation activity. The zinc finger domain (ZFD) specifically recognizes the 5 0 -GGACU-3 0 consensus sequence of RNA, forming an RNA-binding interface. Although METTL14 has the folding configuration similar to METTL13, the cavity where RNA substrates bind is not possessed. 22,23 However, recent research has uncovered the important role of METTL14 in the crosstalk between histone modification and RNA methylation. METTL14 recognizes and directly binds with histone H3 trimethylation at Lys36 (H3K36me3), facilitating MAC to adjacent RNAPII, thereby installing m 6 A in actively transcribed nascent RNAs co-transcriptionally. This mechanism may suggest how m 6 A is specifically deposited in the transcriptome. 24 The discovery of Wilms' tumor 1-associating protein (WTAP) enriched the composition of the m 6 A methyltransferase complex. WTAP exhibits affinity for the methyltransferase complex, locating the METTL3-METTL14 complex at nuclear speckles and recruiting them to mRNA targets, regulating expression and alternative splicing of genes with the assistance of METTL3. 25 Studies focusing on interaction surfaces within the METTL3-METTL14-WTAP complex unveiled the novel overall architecture. In WTAP, the METTL3-binding surface is within the N-terminal 150 aa, while the WTAP interaction surface on METTL3 is an N-terminal helical structure that is necessary and sufficient for WTAP-METTL3 interaction. Strikingly, METTL3 phosphorylation in the WTAP interaction surface does not affect subcellular localization, WTAP interaction, or catalytic activity. Moreover, the C-terminal arginine-glycine repeats (RGG) of METTL14, contributing to RNA substrate binding, are indispensable for MAC catalytic activity. The finding is consistent with previous conclusions about the function of METTL14. 26 More WTAP-related proteins and their relevant regulatory factors have been discovered in further studies. Hitherto, we defined the complex consisting of WTAP, VIRMA, Hakai, RBM15 (RNA-binding motif protein 15), and ZC3H13 (a zinc-finger protein) as MACOM (m 6 A-METTL-associated complex). 11,[27][28][29] Interacting with each other, these factors accumulate around target RNAs and catalyze methylation in specific sites. WTAP plays a central role in MACOM and mediates the nuclear speckle localization of the com-plex with assistance of BCLAF1 and THRAP3 (arginine/serine-rich domain-containing proteins). 30 Recently, METTL16 was regarded as another human m 6 A methyltransferase that targets pre-mRNAs and non-coding RNAs. METTL16 participates in splicing regulation by catalyzing N6-methylation in A43, which is in the specific sequence of U6 small nuclear RNA (snRNA). The alteration in A43 can influence the base pairing at 5 0 splice sites of pre-mRNAs during splicing. 13 Intriguingly, studies also revealed METTL16 as a regulator that maintains SAM homeostasis by binding with MAT2A mRNA hairpins, implicating its likely important role in early development. 31,32 Demethylases Termed as erasers, demethylases can remove m 6 A in RNA. To date, only two demethylases, fat mass and obesity-associated protein (FTO) and AlkB homolog 5 (ALKBH5), have been identified. FTO is the first identified demethylase, which is highly expressed in brain and muscle. 33 FTO mediates demethylation with its oxidative activity targeting the m 6 A-rich region in RNA. 34 The mechanism of sequence-specific m 6 A demethylation was revealed by further studies that demonstrated that FTO and RCas9 can fuse together as an RNAtargeting module. The resulting RCas9-FTO retained demethylation activity and bound to RNA in a sequence-specific manner depending on the single-guide RNA (sgRNA) and PAMmer. 35 Furthermore, consensus motifs GGACU and RRACU were discovered in target sequences. FTO specifically removed m 6 A from GGACU and RRACU motifs in a concentration-dependent manner. 4 Additionally, FTO also mediates the demethylation of cap N6, 2 0 -O-dimethyladenosine (cap m 6 A m ) in snRNA and mRNA. Compared with the demethylation of m 6 A, FTO act differently in m 6 A m demethylation, which can be accounted for by different cellular distribution features of FTO. Experiments conducted in polyadenylated RNAs among different cell lines manifested that FTO is more active with regard to m 6 A m (81.9% of m 6 A m and 20.3% of m 6 A were demethylated in vitro). m 6 A is preferentially demethylated by FTO in nucleus, whereas cap m 6 A m is a prominent target in cytoplasm. 36 The cap m 6 A m at +1 position from 5 0 cap in mRNA confers resistance to mRNA-decapping enzyme DCP2 on transcripts, thereby enhancing mRNA stability. 37 FTO-mediated m 6 A m demethylation was also detected in snRNAs (U1 RNA and U2 RNA; both the cap and internal m 6 A m modifications are more significant in U2 RNA), but m 6 A demethylation only happened in U6 RNA. 36 Demethylation of m 6 A m at the sites adjacent to snRNA cap lead to relatively decreased m 6 A m -snRNA levels. However, the function of FTO can be inhibited by the oncometabolite-2-hydroxyglutarate, resulting in increased m 6 A m -snRNA levels, which may change patterns of alternative splicing. 38 The FTO-induced noteworthy changes of snRNAs indicate that FTO is capable of having an influence on mRNA splicing.
ALKBH5 catalyzes demethylation of m 6 A in RNA and contributes to normal splicing and the formation of longer 3 0 UTR mRNAs. Therefore, ALKBH5 also takes part in maintaining stability of transcripts. 33,39 In light of the similar functions of ALKBH5 and FTO, the underlying mechanism by which the demethylases selectively recognize their target transcripts is intriguing. Some research has indicated that an m 6 A-induced conformational change on RNA may account for the specificity but that the consensus sequence (GG(m 6 A)CU) is not indispensable for the specific recognition. 40 m 6 A-Binding Proteins m 6 A modification exerts biological functions by binding to YTH domain-containing proteins (YTHDC1-2), YTH-family proteins (YTHDF1-3), and other interacting factors. The proteins are collectively defined as readers. In the nucleus, YTHDC1 promotes exon inclusion of targeted mRNAs and regulates mRNA splicing and export from nucleus to cytoplasm by recruiting pre-mRNA splicing factor SFSF3. 41,42 YTHDC1 was also established to participate in the maintenance of intracellular SAM levels, thereby regulating the level of methylation. 32 HNRNPA2B1 is a direct m 6 A reader which takes part in primary RNA (pri-miRNA) processing and alternative splicing by interacting with the DGCR8 protein. 43 In the cytoplasm, YTHDF1-3 proteins work synergistically to influence RNA metabolism. 41,44,45 YTHDF3 promotes translation initiation of its target mRNA with assistance of YTHDF1 and initiation factor eIF4A3. YTHDF3 also mediates mRNA degradation by cooperating with YTHDF2. Similar to YTHDF3, YTHDC2 also has a dual effect on target mRNA. YTHDC2 enhances translation efficiency of target mRNA, while knockdown of YTHDC2 results in an upregulation of m 6 A-modified transcripts. This novel function of YTHDC2 is essential for fertility in mammals and ensures the transition from mitosis to meiosis. 46,47 Especially, transcripts with m 6 A in the 5 0 UTR can be translated by directly binding elF3, independent of YTHDF1 and cap-binding factor eIF4E. In addition, diverse cellular stresses selectively result in an increase in mRNAs with 5 0 UTR m 6 A. 5 As a new class of m 6 A readers discovered recently, IGF2BP1/2/3 (insulin-like growth factor 2 mRNA-binding proteins 1, 2, and 3) recognize the consensus GG(m 6 A)C sequence and bind m 6 A by their K homology domains. In such an m 6 A-dependent manner, IGF2BP1/ 2/3 prevent target mRNAs from degradation and promote mRNA translation. 48 AML is a malignant disease originating from hematopoietic stem cells or progenitor cells. Chemotherapy and hematopoietic stem cell transplantation are general treatments for AML, but targeted therapy, demethylation therapy, and immunotherapy also show promising therapeutic effects. With high expression of methyltransferases, AML cells feature elevated m 6 A, which contributes to the maintenance of multilineage differentiation potential and inhibits cell differentiation in AML. 49,50 METTL3 recruited by CEBPZ promotes translation of SP1 by upregulating the m 6 A level. SP1 subsequently activates the oncogene c-MYC, which can result in the development of AML. 50 Similarly, m 6 A-modified BCL2 and PTEN induced by elevated METTL3 levels in AML can also lead to AML development caused by downregulation of phosphorylated phosphatidylinositol 3-kinase (PI3K)/AKT. 49 However, elevated non-functional METTL3 also activates the PI3K/AKT pathway, which implicated that there are certain mechanisms independent of m 6 A modification. 49 Strikingly, similar to m 6 A readers, METTL3 can also promote the translation of mRNAs by binding m 6 A-modified regions close to the stop codon and interacting with eIF3h. 51 METTL14 is essential for maintaining self-renewal of AML cells, while knockdown of METTL14 promotes myeloid differentiation. The consistent high expression of METTL14 is attributed to the dysregulation of SPI1. 52 Oncogenes MYB and MYC are targets of METTL14. However, YTHDF proteins are not involved in promoting stability and translation of target mRNAs, while knockdown of METTL14 has an effect on the quantity of MYB and MYC mRNA. 52 METTL16 indirectly regulates the activity of METTL3/MELL14 by regulating the expression of MAT2A, which maintains appropriate SAM levels. 32,53 WTAP has been found upregulated in AML and to plays an oncogenic role. 54 Increased expression of WTAP in AML cell lines alters alternative splicing, promotes proliferation, and blocks differentiation. 30,54 Studies found that the increased WTAP protein levels result from simultaneous high levels of cytoplasmic METTL3, in agreement with the conclusion that METTL3 and METTL14 are WTAP complex interactors. 55 The RBM15-MKL1 fusion was originally detected in infants and the transcripts were analyzed for diagnosis of AMKL. 56,57 Interacting with the KMT2G, RBM15-MKL1 fusion protein enhanced cell proliferation. 58 RBM15 can inhibit myeloid differentiation in hematopoietic cells by stimulating Notch signaling via RBPJkappa 59 and contribute to adult hematopoiesis and normal megakaryocyte development mediated at least in part by c-Myc. Therefore, the function of RBM15 in AML is correlated with the activation of Notch signaling and interaction with c-Myc. 60 In addition, overexpression of PRMT1 leads to aberrant alternative splicing and blocks AML cell differentiation by inducing RBM15 ubiquitylation and degradation. 61,62 FTO is overexpressed in AML with MLL rearrangements, PML-RARA, FLT3-ITD, or NPM1 mutations. 63 FTO can decrease m 6 A levels in certain mRNA transcripts, including the tumor suppressor ankyrin repeat and ASB2 (SOCS box protein 2) and RARA (retinoic acid receptor alpha), thereby suppressing the expression of ASB2 and RARA, which regulate normal hematopoiesis and all-trans-retinoic acid (ATRA)-induced AML cell differentiation. Therefore, overexpressed FTO mediates leukemogenesis and indicates better efficacy of ATRA treatment in AML cells. 63 With the capacity for catalyzing demethylation of m 6 A m in mRNA and snRNA and N 1 -methyladenosine (m1A) in tRNA, 36 FTO may mediate oncogenesis independent of m 6 A. Similarly, CAPAM (cap-specific adenosine methyltransferase) mediating m 6 A modification at the 5 0 cap in mRNA may correlate with AML, but the hypothesizes have yet to be identified. 64,65 Recently, an investigation in leukemia patients indicated that genetic alterations of m 6 A regulatory genes were associated with p53 mutations in AML. These alterations always predict a poor survival rate. 66 Genetic aberrations, that is, CBFA2T3/GLIS2 and NUP98/ KDM5A, may be used for risk group stratification of pediatric AMKL and treatment tailoring. 67 Colorectal Cancer YTHDF1 and METTL3 have been identified to play significant roles in colorectal cancer (CRC). YTHDF1 was established to regulate tumorigenesis in CRC cells via the Wnt/b-catenin pathway. Mechanistically, studies found that YTHDF1 induced aberrant activation of Wnt/b-catenin signaling via recognizing and promoting translation of FZD9 and Wnt6 mRNA in an m 6 A-dependent manner. According to clinical and laboratory data, high YTHDF1 expression in patients signaled poorer overall survival, while silencing of YTHDF1 in vitro resulted in poor efficacy of anticancer drugs and suppression of cancer proliferation. However, it is notable that c-Myc was indispensable in the carcinogenic mechanism of YTHDF1, indicating the theory of a c-Myc-driven YTHDF1 axis. 68 Moreover, YTHDF1 gene copy number amplification contributes to YTHDF1 overexpression in CRC. 69 METTL3, acting as a carcinogenic factor, was observed to be overexpressed in CRC metastatic tissues and released a signal of poor prognosis. Confirmed as an m 6 A methyltransferase (as previously mentioned), METTL3 is capable of methylating SRY (sex determining region Y)-box 2 (SOX2), which is essential for maintaining self-renewal. IGF2BP2 subsequently recognizes the methylated SOX2 transcripts, thereby preventing SOX2 mRNA degradation. 70 In addition, studies have shown that lncRNA RP11 triggers the dissemination of CRC cells. RP11 induce cell dissemination by post-translationally regulates the stability of Zeb1, an epithelialmesenchymal transition (EMT)-related transcription factor. An m 6 A-dependent manner is involved in the accumulation of RP11 in nuclear. Mechanistically, the RP11/HNRNPA2B1/mRNA complex prevented the proteasomal degradation of Zeb1 by promoting the mRNA degradation of Siah1 and Fbxo45. 71

Gastric Cancer
The molecular mechanism in the occurrence of gastric cancer (GC) remains unclear, but some conclusions have emerged during studies. Some studies have given way to the idea that METTL3 is a carcinogenic factor of GC. METTL3 was observed to be significantly overexpressed in GC tissues, and the expression level of METTL3 was positively related to the tumor/node/metastasis (TNM) stage of GC. m 6 A methylation level was also upregulated simultaneously. METTL3 leads to inactivation of the AKT signaling pathway, while knockdown of METTL3 activated the apoptotic pathway in GC cells. Downregulation of METTL3 suppressed the proliferation and mobility of GC cells. 72,73 However, another study came to the opposite conclusion that downregulation of m 6 A contributed to GC cell proliferation and invasiveness by activating Wnt and PI3K/AKT signaling, indicating that the m 6 A methyltransferases may suppress tumorigenesis. 74 Moreover, compared with adjacent non-tumor tissues, FTO expression in GC tissues featured a high level in protein and mRNA, suggesting that FTO may play a significant role in GC. Over-expression of FTO promotes the proliferation, migration, and invasion of GC cells and is positively related to TNM stage. 75

Breast Cancer
Aberrant m 6 A level and dysregulation of METTL3, METTL14, WTAP, and FTO have been observed in breast cancer (BC) cells. 76 Decreased m 6 A level was confirmed to mediate abnormal expression of certain tumor suppressor genes in a premature polyadenylation (pPA)-dependent manner. 77 Studies have shown that FTO was upregulated in human BC, promoting proliferation, colony formation, and metastasis of BC cells. Mechanistically, FTO inhibits the function of BNIP3, which acts as a tumor suppressor. FTO mediated m 6 A demethylation at the RRACH motif near the stop codon of BNIP3 mRNA and induced its degradation in an YTHDF2-independent manner. 78 As a reader of the m 6 A, HNRNPA2/B1 is upregulated in BC cells, reducing sensitivity to antagonists of estrogen receptor by regulating levels of specific miRNAs. 79 Specially, some studies found the dual role of m 6 A in BC. In immortalized and oncogenically transformed human mammary epithelial cells (HMECs), m 6 A levels were significantly decreased with a decrease of METTL3 and an increase of ALKBH5. However, increased m 6 A levels in transformed cells, resulting from overexpression of METTL3 and METTL14, or deletion of ALKBH5, promoted proliferation and migration. Interestingly, m 6 A levels in the immortalized and transformed cells were found to increase under stress with constant levels of METTL3, METTL14, and ALKBH5, suggesting other underlying pathways for regulation of m 6 A levels. 80

Lung Cancer
Deregulation of FTO and METTL3 has been detected in lung cancer, which indicated their significant roles in tumorigenesis and development. Studies established that FTO plays an oncogenic role in NSCLC (non-small-cell lung cancer) and LUSC (lung squamous cell carcinoma). In human NSCLC tissues, overexpressed FTO promoted proliferation and colony formation of cancer cells by increasing USP7 mRNA level and USP7 expression. 81 FTO was also involved in the progression of LUSC. Mechanistically, FTO decreased m 6 A levels and mRNA stability in the MZF1 mRNA transcript, thereby promoting expression of MZF1 and mediating carcinogenesis. 82 METTL3-eIF3h enhances translation of oncogenic mRNAs in primary lung tumors and leads to oncogenic transformation. 51 METTL3 is also implicated in NSCLC progression, promoting cell growth and survival via the PI3K/Akt pathway. 83 Additionally, METTL16 was found to bind the 3 0 terminal triple helix of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a cancer-promoting long noncoding RNA, and regulated MALAT1 endocellular accumulation. However, the relationship between tumorigenesis and this mechanism remains unclear. 84 In addition, when researching into the deregulated autophagy in human cancers, aberrant activation of the oncogene UBE2C was noticeable. In NSCLC, upregulated ALKBH5 maintains a lower m 6 A level in UBE2C mature RNAs and activates UBE2C expression, thereby repressing autophagy. Depletion of UBE2C induces attenuated proliferation, clonogenicity, and invasive growth of NSCLC cells. 85

Hepatocellular Carcinoma
Overexpressed METTL3, METTL14 downregulation, and upregulated m 6 A readers have been observed in hepatocellular carcinoma (HCC) cells. Overexpression of METTL3 is a signal of poor prognosis in HCC, and upregulated YTHDF1 is positively correlated with TNM stage, indicating their important roles in HCC cells metastasis and progression. [86][87][88] With a YTHDF2-dependent mechanism, METTL3 mediated m 6 A modification of the SOCS2 suppressor, thereby repressing SOCS2 expression and promoting HCC progression. 86 Contrastingly, METTL14 performs a repressing effect in tumor metastasis by interacting with the microprocessor protein DGCR8 and promoting pre-miRNA126 process via m 6 A modification. 89 VIRMA, another m 6 A writer, was also identified as an oncogenic factor, facilitating migration, proliferation, and invasion of HCC cells by inducing m 6 A modification of ID2 mRNA. 90

Melanoma
Melanoma is a type of malignant cancer that originates from pigment-producing melanocytes. The pathogenesis of melanoma still remains unclear, but some studies have given insight into the mechanism of oncogenesis. FTO and METTL3 are significantly upregulated in human melanoma. 91,92 FTO-mediated m 6 A mRNA demethylation promotes melanoma tumorigenesis and anti-PD-1 resistance. Knockdown of FTO increases m 6 A modification in the critical protumorigenic melanoma cell-intrinsic genes, including PD-1, CXCR4, and SOX10, inducing RNA degradation via binding YTHDF2. In mice, knockdown of FTO markedly enhances mela-noma cell sensitivity to interferon (IFN)-g and anti-PD-1 treatment, which suggested a promising anti-cancer therapy. 91 Overexpressed METTL3 enhances colony formation and invasion of melanoma cells via promoting accumulation of MMP2 and N-cadherin. 92

Glioblastoma (GBM)
Glioblastoma is the most aggressive malignant tumor of central nervous system. Although there are multiple clinical trials for GBM, the recurrence rate is still very high. GB stem cells (GSC) are a subset of GBM cells, closely correlating with tumorigenesis, proliferation, and recurrence. Many studies aiming at GSCs have been performed in recent years. Upregulated m 6 A mRNA modification was identified to induce the suppression of GSC self-renewal and tumorigenesis. Mechanistically, the changing m 6 A level altered mRNA expression of certain genes that play critical roles in GSCs. METTL3 and METTL14 suppress GSC growth and self-renewal by downregulating oncogenes (ADAM19, EPHA3, and KLF4) and upregulating tumor suppressors (CDKN2A, BRCA2, and TP53I11). 93 Further studies have shed light on the function of some enzymes. ALKBH5 was overexpressed in GSCs and promoted FOXM1 expression by demethylating its nascent transcripts. Moreover, a lncRNA antisense to FOXM1 (FOXM1-AS) also participated in the ALKBH5-mediated tumorigenesis. 94 Being involved in RNA processing and oncogenic pathways of GSCs, functions of METTL3 are multiple and complex. METTL3 plays a dominant role in m 6 A modification of GSCs and participates in the expression of GSC-specific actively transcribed genes and alternative splicing. Additionally, METTL3 decreases A-to-I RNA editing by downregulating ADAR and ADARB1, whereas it upregulates editing enzymes APOBEC1 and APOBEC3A to increase C-to-U RNA editing. 95

Advances in the m 6 A Modification on Human Cancers
It is becoming increasingly clear that deregulated m 6 A modification is a key factor in growth, metastasis, and drug resistance of various tumors. Recently, some novel regulatory pathways have been found in cancer cells, which may give insight into potential therapeutic strategies to inhibit cancers. Many enzymes and genes have been identified to play vital roles in the sophisticated system and alter therapeutic efficacy.
METTL3 has been considered to be an oncogenic factor in many human cancers, and studies focusing on blocking METTL3-dependent pathways have been performed. METTL3-depleted pancreatic cancer cells exhibited higher sensitivity to anticancer reagents. 96 miR-600induced METTL3 suppression can inhibit NSCLC progression. 83 In bladder cancer cells, METTL3 and YTHDF1 promote oncogene CDCP1 translation by binding m 6 A in the 3 0 UTR of CDCP1 mRNA. The METTL3-m 6 A-CDCP1 axis has been revealed as a target for treating chemical-induced cancers. 97 Moreover, METTL3 participates in the mutagenesis of p53 proteins, which promotes proliferation, metastasis, and drug resistance of cancer cells. METTL3 catalyzes the formation of m 6 A at the point-mutated codon 273 (G>A) of p53 pre-mRNA, promoting aberrant splicing of p53 pre-mRNA and expression of p53 R273H mutant protein. Furthermore, Figure 1. Methyltransferases, Demethylases, and m 6 A-Binding Proteins in m 6 A Modification m 6 A is catalyzed by writers and removed by erasers (FTO and ALKBH5). MACOM, consisting of WTAP, VIRMA, Hakai, RBM15, and ZC3H13, ensures the location of the METTL3-METTL14 core complex to nuclear speckle. m 6 A modification exerts biological functions by binding to YTHDC1-2, YTHDF1-3, HNRNPA2B1, IGF2BP1-3, eIF3, and eIF4A3. In the nucleus, YTHDC1 and HNRNPA2B1 bind m 6 A and perform multiple functions. In the cytoplasm, YTHDC2, YTHDF1-3, IGF2BP1-3, eIF3, and eIF4A3 induce translation or degradation of transcripts. eIF3 can promote translation by directly binding m 6 A in the 5 0 UTR. upregulated METTL3 expression is partly attributed to glycosphingolipids, which execute function via activating cSrc and b-catenin signaling. 98 Reduction of m 6 A methylation caused by abnormal MAC has been found in about 70% of human endometrial cancer samples, resulting in activation of the AKT pathway by upregulating the negative AKT regulator PHLPP2 and downregulating the positive AKT regulator mTORC2. The alteration of MAC can be caused by METTL14 mutation (hotspot R298P mutation) or reduced expression of METTL3. 99 FTO has been observed significantly upregulated in human cancers, promoting occurrence, proliferation, and migration of cancer cells. These discoveries have conferred an identity as a therapeutic target on FTO. FB23 and FB23-2 are promising small-molecule FTO EMT, a crucial step for tumor metastasis, has been confirmed to involve an m 6 A/YTHDF1-mediated mechanism. YTHDF1 recognizes upregulated m 6 A in coding sequences of Snail (a key transcription factor of EMT), promoting translation of Snail mRNA. 105 Studies focusing on durable neoantigen-specific immunity also uncovered the critical role of YTHDF1. In classical dendritic cells of mice, YTHDF1 interferes with cross-presentation of tumor antigens and cross-priming of CD8 + T cells. YTHDF1 binds the m 6 A-modified transcripts, which encode lysosomal proteases, promoting the translation of lysosomal cathepsins. Conversely, knockdown of YTHDF1 induces suppression of cathepsins and dramatically enhances cross-presentation. Furthermore, experiments in vivo implicated that YTHDF1 is a potential therapeutic target for enhancing efficacy of PD-L1 checkpoint blockade. 106 What may bring breakthroughs for targeted therapy is the discovery of the cell specificity of YTHDF2 function. YTHDF2 is not essential for normal hematopoietic stem cell (HSC) function, whereas in leukemic stem cells, it blocks the apoptosis pathway by regulating expression of tumor necrosis factor receptor superfamily 2 (TNFRSF2). 107 Suppression of YTHDF2 is promising for maintenance of HSC self-renewal capacity after transplantation. Knockdown of human YTHDF2 led to a significant increase of HSCs (an average increase of 14.3-fold and 13.6-fold in the ex vivo expansion of human umbilical cord blood [hUCB] HSCs, a 5.1-fold increase in colonyforming units [CFU], and more than an 8-fold increase in functional hUCB HSCs in the secondary serial of a limiting dilution transplantation assay), and hematopoietic malignancies were not detected in YTHDF2-knockout mice. Mechanically, YTHDF2 promotes decay of m 6 A-modified mRNAs, which are critical for stem cell selfrenewal. 108 Thus, YTHDF2 is identified as a unique therapeutic target.

Conclusion and Perspective
To summarize, overexpression or suppression of writers and erasers lead to aberrant levels of m 6 A in the transcripts of oncogenes or tumor suppressor genes, regulating translation efficiency and degradation with full participation of readers (Table 1). These resulting proteins induce activation of signaling pathways (Wnt/b-catenin, PI3K/ AKT) and then create cascade reactions. However, we know little about how the enzymes specifically recognize the genes and transcripts.
By detecting the expression levels of the enzymes in cancerous tissues and adjacent non-cancerous tissues, studies have shown the prevalent overexpression of METTL3, METTL14, FTO, and others. However, METTL3 was identified as a suppressor in certain cancers, which is in conflict with previous conclusions. Further studies have indicated that the m 6 A level was changing with the development of cancer, which may partly explain the contradiction. The exact regulatory mechanism of m 6 A-related protein expression is not fully clear. m 6 A modification provides a new idea for cancer therapy. Further studies are needed for weakening drug resistance of cancer cells and improving the accuracy of targeted therapy.

AUTHOR CONTRIBUTIONS
L.L., Y.W., and J.W. wrote and drafted the manuscript and figures. H.F., Z.Q., and J.L., revised the manuscript. All authors read and approved the final manuscript.

CONFLICTS OF INTEREST
The authors declare no competing interests.