High-Fat Diet Promotes Colorectal Tumorigenesis Through Modulating Gut Microbiota and Metabolites.

BACKGROUND AND AIMS: Dietary fat intake is associated with increased risk of colorectal cancer (CRC). We examined the role of high-fat diet (HFD) in driving CRC through modulating gut microbiota and metabolites. METHODS: HFD or control diet was fed to mice littermates in CRC mouse models of an azoxymethane (AOM) model and Apcmin/+ model, with or without antibiotics cocktail treatment. Germ-free mice for fecal microbiota transplantation were used for validation. Gut microbiota and metabolites were detected using metagenomic sequencing and high-performance liquid chromatography-mass spectrometry, respectively. Gut barrier function was determined using lipopolysaccharides level and transmission electron microscopy. RESULTS: HFD promoted colorectal tumorigenesis in both AOM-treated mice and Apcmin/+ mice compared with control diet-fed mice. Gut microbiota depletion using antibiotics attenuated colon tumor formation in HFD-fed mice. A significant shift of gut microbiota composition with increased pathogenic bacteria Alistipessp.Marseille-P5997 and Alistipessp.5CPEGH6, and depleted probiotic Parabacteroides distasonis, along with impaired gut barrier function was exhibited in HFD-fed mice. Moreover, HFD-modulated gut microbiota promotes colorectal tumorigenesis in AOM-treated germ-free mice, indicating gut microbiota was essential in HFD-associated colorectal tumorigenesis. Gut metabolites alteration, including elevated lysophosphatidic acid, which was confirmed to promote CRC cell proliferation and impair cell junction, was also observed in HFD-fed mice. Moreover, transfer of stools from HFD-fed mice to germ-free mice without interference increased colonic cell proliferation, impaired gut barrier function, and induced oncogenic genes expression. CONCLUSIONS: HFD drives colorectal tumorigenesis through inducing gut microbial dysbiosis, metabolomic dysregulation with elevated lysophosphatidic acid, and gut barrier dysfunction in mice.

Squalene epoxidase drives cancer cell proliferation and promotes gut dysbiosis to accelerate colorectal carcinogenesis.

OBJECTIVE: Aberrant lipid metabolism is a hallmark of colorectal cancer (CRC). Squalene epoxidase (SQLE), a rate-limiting enzyme in cholesterol biosynthesis, is upregulated in CRC. Here, we aim to determine oncogenic function of SQLE and its interplay with gut microbiota in promoting colorectal tumourigenesis. DESIGN: Paired adjacent normal tissues and CRC from two cohorts were analysed (n=202). Colon-specific Sqle transgenic (Sqle tg) mice were generated by crossing Rosa26-lsl-Sqle mice to Cdx2-Cre mice. Stools were collected for metagenomic and metabolomic analyses. RESULTS: SQLE messenger RNA and protein expression was upregulated in CRC (p<0.01) and predict poor survival of patients with CRC. SQLE promoted CRC cell proliferation by inducing cell cycle progression and suppressing apoptosis. In azoxymethane-induced CRC model, Sqle tg mice showed increased tumourigenesis compared with wild-type mice (p<0.01). Integrative metagenomic and metabolomic analyses unveiled gut dysbiosis in Sqle tg mice with enriched pathogenic bacteria, which was correlated to increased secondary bile acids. Consistent with detrimental effect of secondary bile acids, gut barrier function was impaired in Sqle tg mice, with reduced tight junction proteins Jam-c and occludin. Transplantation of Sqle tg mice stool to germ-free mice impaired gut barrier function and stimulated cell proliferation compared with control mice stool. Finally, we demonstrated that terbinafine, a SQLE inhibitor, could be repurposed for CRC by synergising with oxaliplatin and 5-fluorouracil to inhibit CRC growth. CONCLUSION: This study demonstrates that SQLE mediates oncogenesis via cell intrinsic effects and modulation of gut microbiota-metabolite axis. SQLE represents a therapeutic target and prognostic marker in CRC.

Cigarette smoke promotes colorectal cancer through modulation of gut microbiota and related metabolites.

OBJECTIVE: Cigarette smoking is a major risk factor for colorectal cancer (CRC). We aimed to investigate whether cigarette smoke promotes CRC by altering the gut microbiota and related metabolites. DESIGN: Azoxymethane-treated C57BL/6 mice were exposed to cigarette smoke or clean air 2 hours per day for 28 weeks. Shotgun metagenomic sequencing and liquid chromatography mass spectrometry were parallelly performed on mice stools to investigate alterations in microbiota and metabolites. Germ-free mice were transplanted with stools from smoke-exposed and smoke-free control mice. RESULTS: Mice exposed to cigarette smoke had significantly increased tumour incidence and cellular proliferation compared with smoke-free control mice. Gut microbial dysbiosis was observed in smoke-exposed mice with significant differential abundance of bacterial species including the enrichment of Eggerthella lenta and depletion of Parabacteroides distasonis and Lactobacillus spp. Metabolomic analysis showed increased bile acid metabolites, especially taurodeoxycholic acid (TDCA) in the colon of smoke-exposed mice. We found that E. lenta had the most positive correlation with TDCA in smoke-exposed mice. Moreover, smoke-exposed mice manifested enhanced oncogenic MAPK/ERK (mitogen-activated protein kinase/extracellular signal­regulated protein kinase 1/2) signalling (a downstream target of TDCA) and impaired gut barrier function. Furthermore, germ-free mice transplanted with stools from smoke-exposed mice (GF-AOMS) had increased colonocyte proliferation. Similarly, GF-AOMS showed increased abundances of gut E. lenta and TDCA, activated MAPK/ERK pathway and impaired gut barrier in colonic epithelium. CONCLUSION: The gut microbiota dysbiosis induced by cigarette smoke plays a protumourigenic role in CRC. The smoke-induced gut microbiota dysbiosis altered gut metabolites and impaired gut barrier function, which could activate oncogenic MAPK/ERK signalling in colonic epithelium.

Squalene Epoxidase Induces Nonalcoholic Steatohepatitis Via Binding to Carbonic Anhydrase III and is a Therapeutic Target.

BACKGROUNDS & AIMS: Squalene epoxidase (SQLE) is the rate-limiting enzyme for cholesterol biosynthesis. We elucidated the functional significance, molecular mechanisms, and clinical impact of SQLE in nonalcoholic steatohepatitis (NASH). METHODS: We performed studies with hepatocyte-specific Sqle overexpression transgenic (Sqle tg) mice and mice given high-fat high-cholesterol (HFHC) or methionine- and choline-deficient (MCD) diet to induce NASH. SQLE downstream target carbonic anhydrase III (CA3) was identified using co-immunoprecipitation and Western Blot. Some mice were given SQLE inhibitor (terbinafine) and CA3 inhibitor (acetazolamide) to study the therapeutic effects in NASH. Human samples (N = 217) including 65 steatoses, 80 NASH, and 72 healthy controls were analyzed for SQLE levels in liver tissue and in serum. RESULTS: SQLE is highly up-regulated in human NASH and mouse models of NASH. Sqle tg mice triggered spontaneous insulin resistance, hepatic steatosis, liver injury, and accelerated HFHC or MCD diet-induced NASH development. Mechanistically, SQLE tg mice caused hepatic cholesterol accumulation, thereby triggering proinflammatory nuclear factor-κB signaling and steatohepatitis. SQLE directly bound to CA3, which induced sterol regulatory element-binding protein 1C activation, acetyl-CoA carboxylase, fatty acid synthase, and stearoyl-CoA desaturase1 expression and de novo hepatic lipogenesis. Combined targeting SQLE (terbinafine) and CA3 (acetazolamide) synergistically ameliorated NASH in mice with superior efficacy to either drug alone. Serum SQLE with CA3 could distinguish patients with NASH from steatosis and healthy controls (area under the receiver operating characteristic curve, 0.815; 95% confidence interval, 0.758-0.871). CONCLUSIONS: SQLE drives the initiation and progression of NASH through inducing cholesterol biosynthesis, and SQLE/CA3 axis-mediated lipogenesis. Combined targeting of SQLE and CA3 confers therapeutic benefit in NASH. Serum SQLE and CA3 are novel biomarkers for the noninvasive diagnosis of patients with NASH.

RNA N6-Methyladenosine Methyltransferase METTL3 Facilitates Colorectal Cancer by Activating the m6A-GLUT1-mTORC1 Axis and Is a Therapeutic Target.

BACKGROUND & AIMS: RNA N6-methyladenosine (m6A) modification has recently emerged as a new regulatory mechanism in cancer progression. We aimed to explore the role of the m6A regulatory enzyme METTL3 in colorectal cancer (CRC) pathogenesis and its potential as a therapeutic target. METHODS: The expression and clinical implication of METTL3 were investigated in multiple human CRC cohorts. The underlying mechanisms of METTL3 in CRC were investigated by integrative m6A sequencing, RNA sequencing, and ribosome profiling analyses. The efficacy of targeting METTL3 in CRC treatment was elucidated in CRC cell lines, patient-derived CRC organoids, and Mettl3-knockout mouse models. RESULTS: Using targeted clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 dropout screening, we identified METTL3 as the top essential m6A regulatory enzyme in CRC. METTL3 was overexpressed in 62.2% (79/127) and 88.0% (44/50) of primary CRCs from 2 independent cohorts. High METTL3 expression predicted poor survival in patients with CRC (n = 374, P < .01). Functionally, silencing METTL3 suppressed tumorigenesis in CRC cells, human-derived primary CRC organoids, and Mettl3-knockout mouse models. We discovered the novel functional m6A methyltransferase domain of METTL3 in CRC cells by domain-focused CRISPR screening and mutagenesis assays. Mechanistically, METTL3 directly induced the m6A-GLUT1-mTORC1 axis as identified by integrated m6A sequencing, RNA sequencing, ribosome sequencing, and functional validation. METTL3 induced GLUT1 translation in an m6A-dependent manner, which subsequently promoted glucose uptake and lactate production, leading to the activation of mTORC1 signaling and CRC development. Furthermore, inhibition of mTORC1 potentiated the anticancer effect of METTL3 silencing in CRC patient-derived organoids and METTL3 transgenic mouse models. CONCLUSIONS: METTL3 promotes CRC by activating the m6A-GLUT1-mTORC1 axis. METTL3 is a promising therapeutic target for the treatment of CRC.

Prostaglandin E2-Mediated Impairment of Innate Immune Response to A(H1N1)pdm09 Infection in Diet-Induced Obese Mice Could Be Restored by Paracetamol.

BACKGROUND: Obesity is associated with increased severity of influenza infection. However, the underlying mechanism is largely unknown. METHODS: We employed a mouse model with diet-induced obesity (DIO) to study the innate immune responses induced by influenza virus. RESULTS: The lungs of DIO mice were heavily affected by obesity-associated chronic systemic inflammation with a significant increase in inflammatory cytokines/chemokines. Concurrently, lipid immune mediator prostaglandin E2 (PGE2) was also significantly elevated in DIO mice. However, the DIO mice mounted a blunted and delayed upregulation of mRNA and protein concentrations of interferon-ß and inflammatory cytokines/chemokines upon A(H1N1)pdm09 virus (H1N1/415742Md) challenge compared with those of lean mice. PGE2 concentrations were significantly higher in the lungs of DIO mice compared to that of lean mice postchallenge. Treatment with paracetamol in challenged DIO mice significantly enhanced the expression of interferon-α/ß and cytokine genes at days 1 and 3 postinfection compared with that of untreated DIO mice. Furthermore, paracetamol treatment alone started 3 days before virus challenge and continued until 6 days postchallenge ameliorated the severity of a lethal H1N1/415742Md infection in DIO mice with improved survival. CONCLUSIONS: Impaired innate response to influenza in DIO mice is associated with elevated PGE2, which could be restored to some degree by paracetamol treatment.

Middle East Respiratory Syndrome Coronavirus Efficiently Infects Human Primary T Lymphocytes and Activates the Extrinsic and Intrinsic Apoptosis Pathways.

Middle East respiratory syndrome (MERS) is associated with a mortality rate of >35%. We previously showed that MERS coronavirus (MERS-CoV) could infect human macrophages and dendritic cells and induce cytokine dysregulation. Here, we further investigated the interplay between human primary T cells and MERS-CoV in disease pathogenesis. Importantly, our results suggested that MERS-CoV efficiently infected T cells from the peripheral blood and from human lymphoid organs, including the spleen and the tonsil. We further demonstrated that MERS-CoV infection induced apoptosis in T cells, which involved the activation of both the extrinsic and intrinsic apoptosis pathways. Remarkably, immunostaining of spleen sections from MERS-CoV-infected common marmosets demonstrated the presence of viral nucleoprotein in their CD3(+) T cells. Overall, our results suggested that the unusual capacity of MERS-CoV to infect T cells and induce apoptosis might partly contribute to the high pathogenicity of the virus.

Recombinant influenza A virus hemagglutinin HA2 subunit protects mice against influenza A(H7N9) virus infection.

A novel avian influenza A(H7N9) virus has emerged to infect humans in eastern China since 2013. An effective vaccine is needed because of the high mortality despite antiviral treatment and intensive care. We sought to develop an effective vaccine for A(H7N9) virus. The HA2 subunit was chosen as the vaccine antigen because it is highly conserved among the human A(H7N9) virus strains. Moreover, in silico analysis predicted two immunogenic regions within the HA2 subunit that may contain potential human B-cell epitopes. The HA2 fragment was readily expressed in Escherichia coli. In BALB/c mice, intraperitoneal immunization with two doses of HA2 with imiquimod (2-dose-imiquimod) elicited the highest geometric mean titer (GMT) of anti-HA2 IgG (12699), which was greater than that of two doses of HA2 without imiquimod (2-dose-no-adjuvant) (6350), one dose of HA2 with imiquimod (1-dose-imiquimod) (2000) and one dose of HA2 without imiquimod (1-dose-no-adjuvant) (794). The titer of anti-HA2 IgG was significantly higher in the 1-dose-imiquimod group than the 1-dose-no-adjuvant group. Although both hemagglutination inhibition titers and microneutralization titers were below 10, serum from immunized mice showed neutralizing activity in a fluorescent focus microneutralization assay. In a viral challenge experiment, the 2-dose-imiquimod group had the best survival rate (100 %), followed by the 2-dose-no-adjuvant group (90 %), the 1-dose-imiquimod group (70 %) and the 1-dose-no-adjuvant group (40 %). The 2-dose-imiquimod group also had significantly lower mean pulmonary viral loads than the 1-dose-imiquimod, 1-dose-no-adjuvant and non-immunized groups. This recombinant A(H7N9)-HA2 vaccine should be investigated as a complement to egg- or cell-based live attenuated or subunit influenza vaccines.

A cholesterol tag at the N terminus of the relatively broad-spectrum fusion inhibitory peptide targets an earlier stage of fusion glycoprotein activation and increases the peptide's antiviral potency in vivo.

In previous work, we designed peptides that showed potent inhibition of Newcastle disease virus (NDV) and infectious bronchitis virus (IBV) infections in chicken embryos. In this study, we demonstrate that peptides modified with cholesterol or 3 U of polyethylene glycol (PEG3) conjugated to the peptides' N termini showed even more promising antiviral activities when tested in animal models. Both cholesterol- and cholesterol-PEG3-tagged peptides were able to protect chicken embryos from infection with different serotypes of NDV and IBV when administered 12 h prior to virus inoculation. In comparison, the untagged peptides required intervention closer to the time of viral inoculation to achieve a similar level of protection. Intramuscular injection of cholesterol-tagged peptide at 1.6 mg/kg 1 day before virus infection and then three times at 3-day intervals after viral inoculation protected 70% of the chickens from NDV infection. We further demonstrate that the cholesterol-tagged peptide has an in vivo half-life greater than that of untagged peptides. It also has the potential to cross the blood-brain barrier to enter the avian central nervous system (CNS). Finally, we show that the cholesterol-tagged peptide could play a role before the viral fusion peptide's insertion into the host cell and thereby target an earlier stage of fusion glycoprotein activation. Our findings are of importance for the further development of antivirals with broad-spectrum protective effects.

Tannins amount determines whether tannase-containing bacteria are probiotic or pathogenic in IBD.

The role of dietary tannin in inflammatory bowel disease (IBD) is still not clear. Therefore, we aim to study the effect of TA in the progression of IBD. Dextran sulphate sodium (DSS)-induced model was used to mimic IBD. Metagenomics and metabolomics were performed to study the alteration of intestinal microbiota and metabolites. NCM460 and THP-1 cells were used for in vitro study. The amount of TA was associated with the outcomes of DSS-induced IBD as evidenced by in vivo and in vitro studies. Metabolomic and metagenomic analyses revealed that TA-induced enrichment of microbial metabolite gallic acid (GA) was responsible for the action of TA. Mechanistically, protective dose of GA promoted colonic mucus secretion to suppress bacterial infection and that it ameliorated DSS-induced epithelial damage by inhibiting p53 signaling, whereas toxic dose of GA directly caused epithelial damage by promoting cell cycle arrest. Therapeutic experiment showed protective dose of GA-promoted recovery of DSS-induced colonic inflammation. The role of tannase-containing bacteria can be transformed under different conditions in IBD progression.

Mouse Models for Application in Colorectal Cancer: Understanding the Pathogenesis and Relevance to the Human Condition.

Colorectal cancer (CRC) is a malignant disease that is the second most common cancer worldwide. CRC arises from the complex interactions among a variety of genetic and environmental factors. To understand the mechanism of colon tumorigenesis, preclinical studies have developed various mouse models including carcinogen-induced and transgenic mice to recapitulate CRC in humans. Using these mouse models, scientific breakthroughs have been made on the understanding of the pathogenesis of this complex disease. Moreover, the availability of transgenic knock-in or knock-out mice further increases the potential of CRC mouse models. In this review, the overall features of carcinogen-induced (focusing on azoxymethane and azoxymethane/dextran sulfate sodium) and transgenic (focusing on ApcMin/+) mouse models, as well as their mechanisms to induce colon tumorigenesis, are explored. We also discuss limitations of these mouse models and their applications in the evaluation and study of drugs and treatment regimens against CRC. Through these mouse models, a better understanding of colon tumorigenesis can be achieved, thereby facilitating the discovery of novel therapeutic strategies against CRC.

Capsaicin regulates dyslipidemia by altering the composition of bile acids in germ-free mice.

The improvement of lipid metabolism by capsaicin (CAP) has been extensively studied, mostly with respect to the vanilloid type 1 (TRPV1) ion channel and intestinal flora. In this study, a model was established in germ-free mice by using resiniferatoxin (RTX) to ablate TRPV1 ion channels. Bile acid composition, blood parameters, and colonic transcriptome analyses revealed that CAP could improve dyslipidemia caused by high-fat diet even in the absence of TRPV1 ion channels and intestinal flora. CAP fed to germ mice decreased the concentrations of low-density lipoprotein cholesterol (LDL-C), triglycerides (TG), fasting blood glucose and fasting insulin, increased the concentration of high-density lipoprotein (HDL-C), and decreased the levels of plasma endotoxin and pro-inflammatory factor interleukin 6 (IL-6). Furthermore, CAP could affect both classical and alternative pathways of cholesterol conversion by changing the composition of bile acids, reducing the concentrations of glycocholic acid (GCA), ursodeoxycholic acid (UDCA) and glycochenodeoxycholic acid (GCDCA). First, changing the composition of bile acids inhibited the expression of colon Fgf15. CAP promoted the expression of Cyp7a1 (Cytochrome p450, family 7, subfamily a, and polypeptide 1) in the liver, and thus reduced TC and TG levels. In addition, it could change the composition of bile acids and increase the expression of Cyp7b1 (Cytochrome p450, family 7, subfamily b, and polypeptide 1) in the colon, increase Cyp7b1 protein in the liver and thus inhibit fat accumulation. In conclusion, CAP could alter the composition of bile acids and promote the conversion of cholesterol to bile acids, thereby improving lipid metabolism abnormalities caused by a high-fat diet.

ZNF545 loss promotes ribosome biogenesis and protein translation to initiate colorectal tumorigenesis in mice.

Ribosome biogenesis plays a pivotal role in tumorigenesis by supporting robust protein translation. We investigate the functional and molecular mechanism of Zinc finger protein 545 (ZNF545), a transcriptional repressor for ribosomal RNA (rRNA), in colorectal cancer (CRC). ZNF545 was silenced in CRC compared to adjacent normal tissues (P < 0.0001), implying a tumor-suppressive role. Colon-specific Znf545 knockout in mice accelerated CRC in ApcMin/+ and azoxymethane/dextran sulfate sodium-induced CRC. Mechanistically, we demonstrated that ZNF545 uses its two zinc finger clusters to bind to minimal rDNA promoter, where it assembled transcriptional repressor complex by interacting with KAP1. Znf545 deletion in mouse embryonic fibroblasts not only increased rRNA transcription rate and the nucleolar size and number but also altered the nucleolar composition and architecture with an increased number of fibrillar centers surrounded by net-like dense fibrillar components. Consequently, Znf545 deletion promoted the gene expression of translation machinery, protein translation, and cell growth. Consistent with its tumor-suppressive role, ZNF545 overexpression in CRC cells induced growth arrest and apoptosis. Finally, administration of rRNA synthesis inhibitor, CX-5461, inhibited CRC development in Znf545Δ/ΔApcMin/+ mice. In conclusion, ZNF545 suppresses CRC through repressing rRNA transcription and protein translation. Targeting rRNA biosynthesis in ZNF545-silenced tumors is a potential therapeutic strategy for CRC.

Antifungal Treatment Aggravates Sepsis through the Elimination of Intestinal Fungi.

Prophylactic antifungal therapy is widely adopted clinically for critical patients and effective in reducing the morbidity of invasive fungal infection and improves outcomes of those diagnosed patients; however, it is not associated with higher overall survival. As intestinal commensal fungi play a fundamental role in the host immune response in health and disease, we propose that antifungal therapy may eliminate intestinal fungi and aggravate another critical syndrome, sepsis. Here, with murine sepsis model, we found that antifungal therapy with fluconazole dismissed intestinal fungal burden and aggravated endotoxin-induced but no gram-positive bacteria-induced sepsis. Nevertheless, antifungal therapy did not exert its detrimental effect on germ-free mice. Moreover, colonizing more commensal fungi in the mouse intestine or administration of fungal cell wall component mannan protected the mice from endotoxin-induced sepsis. On the molecular level, we demonstrated that antifungal therapy aggravated endotoxin sepsis through promoting Gasdermin D cleavage in the distal small intestine. Intestinal colonization with commensal fungi inhibited Gasdermin D cleavage in response to lipopolysaccharide challenge. These findings show that intestinal fungi inhibit Gasdermin D-mediated pyroptosis and protect the mice from endotoxin-induced sepsis. This study demonstrates the protective role of intestinal fungi in the pathogenesis of endotoxin-induced sepsis in the laboratory. It will undoubtedly prompt us to study the relationship between antifungal therapy and sepsis in critical patients who are susceptible to endotoxin-induced sepsis in the future.

Microtubule associated protein 9 inhibits liver tumorigenesis by suppressing ERCC3.

BACKGROUND: Chromosomal instability plays an important part in cancer, but its genetic basis in liver tumorigenesis remains largely unclear. We aimed to characterize the mechanistic significance and clinical implication of mitotic regulator microtubule-associated protein 9 (MAP9) in hepatocellular carcinoma (HCC). METHODS: The biological functions of MAP9 were determined by in vitro tumorigenicity assays. Systematic MAP9 knockout mouse (MAP9∆/∆) and hepatocyte-specific MAP9 knockout mouse (MAP9∆/∆hep) were generated to confirm the role of MAP9 in HCC. The clinical impact of MAP9 was assessed in primary HCC tissue samples. FINDINGS: We found that MAP9 was frequently silenced in HCC tissue samples. The transcriptional silence of MAP9 in liver cancer cell lines and tissue samples was mediated by its promoter hypermethylation. MAP9 promoter hypermethylation or downregulation was associated with poor survival and recurrence in patients with HCC. Mechanistically, ectopic expression of MAP9 in LO2 and HepG2 cell lines impaired cell proliferation, colony formation, migration and invasion, and induced cell apoptosis and cycle arrest, whereas knockdown of MAP9 in Miha cell line showed the opposite effects. We found that MAP9∆/∆ mice spontaneously developed a liver hyperplastic nodule and MAP9∆/∆hep accelerated diethylnitrosamine-induced HCC formation. The tumour suppressive effect of MAP9 in HCC was mediated by downregulating excision repair cross-complementation group 3 (ERCC3), a nucleotide excision repair gene. Restoration of ERCC3 expression possessed an oncogenic potency and abrogated the tumour suppressive effects of MAP9. INTERPRETATION: MAP9 is a novel tumour suppressor in HCC by inhibiting ERCC3 expression, and serves as a prognostic factor in HCC patients.

MAP9 Loss Triggers Chromosomal Instability, Initiates Colorectal Tumorigenesis, and Is Associated with Poor Survival of Patients with Colorectal Cancer.

PURPOSE: Chromosomal instability (CIN) is a common phenomenon in colorectal cancer, but its role and underlying cause remain unknown. We have identified that mitotic regulator microtubule-associated protein 9 (MAP9) is a critical regulator of CIN in colorectal cancer. We thus studied the effect of MAP9 loss on colorectal cancer in Map9-knockout mice and in cell lines. EXPERIMENTAL DESIGN: We generated colon epithelial-specific Map9-knockout mice and evaluated colorectal cancer development. Effect of Map9 knockout on colorectal cancer progression was determined in chemical or ApcMin /+ -induced colorectal cancer. Molecular mechanism of MAP9 was determined using spectral karyotyping, microtubule assays, and whole-genome sequencing (WGS). Clinical significance of MAP9 was examined in 141 patients with CRC. RESULTS: Spontaneous colonic tumors (9.1%) were developed in colon epithelium-specific Map9-knockout mice at 17 months, but none was observed in wild-type littermates. Map9 deletion accelerated colorectal cancer formation both in ApcMin /+ mice and azoxymethane-treated mice, and reduced survival in ApcMin /+ mice. Mechanistically, MAP9 stabilized microtubules and mediated mitotic spindle assembly. MAP9 also maintained the spindle pole integrity and protected K-fiber from depolymerization at spindle poles. MAP9 loss induced severe mitosis failure, chromosome segregation errors, and aneuploidy, leading to transformation of normal colon epithelial cells. WGS confirmed enhanced CIN in intestinal tumors from Map9 knockout ApcMin /+ mice. In patients with colorectal cancer, MAP9 was frequently silenced and its downregulation was associated with poor survival. CONCLUSIONS: MAP9 is a microtubule stabilizer that contributes to spindle stability and inhibits colorectal tumorigenesis, supporting the role of MAP9 as a tumor suppressor for preventing CIN in colorectal cancer.

TRIM67 Activates p53 to Suppress Colorectal Cancer Initiation and Progression.

Tripartite motif (TRIM) family proteins participate in a variety of important cellular processes, including apoptosis, cell-cycle arrest, DNA repair, and senescence. In this study, we demonstrated that a novel TRIM family member, TRIM67, was commonly silenced in colorectal cancer and its downregulation was associated with poor survival. Trim67 knockout in ApcMin/+ mice increased the incidence, multiplicity, and burden of colorectal tumors. Similarly, colon-specific knockout of Trim67 significantly accelerated azoxymethane-induced colorectal cancer in mice. RNA sequencing revealed that the antitumor effect of TRIM67 was mediated by activation of the p53 signaling pathway. TRIM67 interacted directly with the C-terminus of p53, inhibiting p53 degradation by its ubiquitin ligase MDM2. TRIM67 was also a transcriptional target of p53; upon cellular stress, p53 bound to the TRIM67 promoter and induced significant upregulation of TRIM67, thereby forming a TRIM67/p53 self-amplifying loop that boosts p53-induced cell growth inhibition and apoptosis. Consequently, loss of this p53-positive regulatory program profoundly compromised p53-mediated responses to chemotherapy-induced DNA damage. Dampened p53 response was also observed in tumors of Trim67 knockout mice and Trim67 knockout embryonic fibroblasts. TRIM67 reactivation restored p53 activation and sensitized colorectal cancer cells to chemotherapy in vitro and in vivo. TRIM67 thus functions as a pivotal tumor suppressor in colorectal cancer and is a potential target for improving chemotherapy responsiveness. SIGNIFICANCE: The TRIM67/p53 axis represents a novel therapeutic target that could be harnessed to improve chemotherapy efficacy in colorectal cancer expressing wild-type p53 but with repressed p53 signaling.

Squalene epoxidase drives NAFLD-induced hepatocellular carcinoma and is a pharmaceutical target.

Nonalcoholic fatty liver disease (NAFLD)-induced hepatocellular carcinoma (HCC) is an emerging malignancy in the developed world; however, mechanisms that contribute to its formation are largely unknown, and targeted therapy is currently not available. Our RNA sequencing analysis of NAFLD-HCC samples revealed squalene epoxidase (SQLE) as the top outlier metabolic gene overexpressed in NAFLD-HCC patients. Hepatocyte-specific Sqle transgenic expression in mice accelerated the development of high-fat, high-cholesterol diet-induced HCC. SQLE exerts its oncogenic effect via its metabolites, cholesteryl ester and nicotinamide adenine dinucleotide phosphate (NADP+). Increased SQLE expression promotes the biosynthesis of cholesteryl ester, which induces NAFLD-HCC cell growth. SQLE increased the NADP+/NADPH (reduced form of NADP+) ratio, which triggered a cascade of events involving oxidative stress-induced DNA methyltransferase 3A (DNMT3A) expression, DNMT3A-mediated epigenetic silencing of PTEN, and activation of AKT-mTOR (mammalian target of rapamycin). In human NAFLD-HCC and HCC, SQLE is overexpressed and its expression is associated with poor patient outcomes. Terbinafine, a U.S. Food and Drug Administration-approved antifungal drug targeting SQLE, markedly inhibited SQLE-induced NAFLD-HCC cell growth in NAFLD-HCC and HCC cells and attenuated tumor development in xenograft models and in Sqle transgenic mice. Suppression of tumor growth by terbinafine is associated with decreased cholesteryl ester concentrations, restoration of PTEN expression, and inhibition of AKT-mTOR, consistent with blockade of SQLE function. Collectively, we established SQLE as an oncogene in NAFLD-HCC and propose that repurposing SQLE inhibitors may be a promising approach for the prevention and treatment of NAFLD-HCC.

Suboptimal Humoral Immune Response against Influenza A(H7N9) Virus Is Related to Its Internal Genes.

Influenza A(H7N9) virus pneumonia is associated with a high case fatality rate in humans. Multiple viral factors have been postulated to account for the high virulence of the virus. It has been reported that patients with influenza A(H7N9) virus infection have relatively low titers of neutralizing antibodies compared to those with seasonal influenza virus infections. In this study, we compared serum hemagglutination inhibition (HI) and microneutralization (MN) antibody titers of mice challenged with wild-type A(H7N9) viruses [H7N9(Anhui) and H7N9(Zhejiang)], an A(H1N1)pdm09 virus [pH1N1(2009)], and a recombinant A(H7N9) virus with PR8/H1N1 internal genes (rg-PR8-H7-N9). All mice infected by H7N9(Anhui) and H7N9(Zhejiang) developed serum HI antibodies at 14 days postinfection (dpi) but no detectable MN antibodies, even at 28 dpi. A low level of neutralizing activity was detected in H7N9(Anhui)- and H7N9(Zhejiang)-infected mice using fluorescent focus MN assay, but convalescent-phase serum samples obtained from H7N9(Anhui)-infected mice did not reduce the mortality of naive mice after homologous virus challenge. Reinfection with homologous A(H7N9) virus induced higher HI and MN titers than first infection. In contrast, pH1N1(2009) virus infection induced robust HI and MN antibody responses, even during the first infection. Moreover, rg-PR8-H7-N9 induced significantly higher HI and MN antibody titers than H7N9(Zhejiang). In conclusion, the internal genes of A(H7N9) virus can affect the humoral immune response against homologous viral surface proteins, which may also contribute to the virulence of A(H7N9) virus.

Toll-like receptor 7 agonist imiquimod in combination with influenza vaccine expedites and augments humoral immune responses against influenza A(H1N1)pdm09 virus infection in BALB/c mice.

Toll-like receptors (TLRs) of the innate immune system are known targets for enhancing vaccine efficacy. We investigated whether imiquimod, a synthetic TLR7 agonist, can expedite the immune response against influenza virus infection when combined with influenza vaccine. BALB/c mice were immunized intraperitoneally with monovalent A(H1N1)pdm09 vaccine combined with imiquimod (VCI) prior to intranasal inoculation with a lethal dose of mouse-adapted A(H1N1)pdm09 virus. For mice immunized 3 days before infection, the survival rates were significantly higher in the VCI group (60%, mean survival time[MST], 11 days) than in the vaccine-alone (30%; MST, 8.8 days), imiquimod-alone (5%; MST, 8.4 days), and phosphate-buffered saline (PBS) (0%; MST, 6.2 days) groups (P < 0.01). In the VCI group, 45 and 35% of the mice survived even when they were infected 2 days or 1 day after immunization. Virus-specific serum IgM, IgG, and neutralizing antibodies appeared earlier with higher geometric mean titers in the VCI group than in the control groups. The pulmonary viral load was significantly lower at all time points postinfection in the VCI, vaccine-alone, and imiquimod-alone groups than in the PBS control group (P < 0.05). The protection induced by VCI was specific for A(H1N1)pdm09 virus but not for A(H5N1) virus. Since imiquimod combined with RNase-treated vaccine is as protective as imiquimod combined with untreated vaccine, mechanisms other than TLR7 may operate in expediting and augmenting immune protection. Moreover, increased gamma interferon mRNA expression and IgG isotype switching, which are markers of the Th1 response induced by imiquimod, were not apparent in our mouse model. The mechanisms of imiquimod-induced immune protection deserve further study.