Lipopolysaccharide-producing Veillonella infantium and Escherichia fergusonii cause vagus nerve-mediated cognitive impairment in mice.

Gut microbiota communicates bidirectionally with the brain through the nervous, immune, and endocrine systems of the gut. In our preliminary study, the fecal microbiota of volunteers with mild cognitive impairment (Fmci) exhibited a higher abundance of Escherichia fergusonii (NK2001), Veillonella infantium (NK2002), and Enterococcus faecium (NK2003) populations compared with those of healthy volunteers. Therefore, we examined the effects of Fmci, NK2001 (gram-negative), NK2002 (gram-negative-like), and NK2003 (gram-positive) on cognitive impairment-like behavior, neuroinflammation, and colitis in mice with or without antibiotics. Fmci transplantation increased cognitive impairment-like behavior, hippocampal tumor necrosis factor (TNF)-α expression, and the size of toll-like receptor (TLR)4+Iba1+, TLR2+Iba1+, and NF-κB+Iba1+ cell populations independent of antibiotic treatment. Oral gavage of NK2001, NK2002, or NK2003, which induced TNF-α expression in Caco-2 cells, significantly increased cognitive impairment-like behavior and hippocampal TNF-α expression and Iba1-positive cell populations and decreased brain-derived neurotrophic factor (BDNF) expression in mice. Celiac vagotomy significantly decreased NK2001- or NK2002-induced cognitive impairment-like behavior and hippocampal Iba1+ cell population and TNF-α expression and increased NK2001- or NK2002-suppressed hippocampal BDNF expression. However, NK2003-induced cognitive impairment-like behavior and hippocampal Iba1+ cell population and TNF-α expression were partially, but not significantly, attenuated by celiac vagotomy. Furthermore, celiac vagotomy did not affect NK2001-, NK2002-, or NK2003-induced lipopolysaccharide (LPS) levels in the blood and feces and TNF-α expression and NF-κB-positive cell population in the colon. In conclusion, LPS-producing NK2001 and NK2002 and LPS-nonproducing NK2003 may induce NF-κB-mediated neuroinflammation through the translocation of byproducts such as LPS and peptidoglycan into the brain through gut-blood/vagus nerve-brain and gut-blood-brain pathways, respectively, resulting in cognitive impairment.

Bifidobacterium bifidum and Lactobacillus paracasei alleviate sarcopenia and cognitive impairment in aged mice by regulating gut microbiota-mediated AKT, NF-κB, and FOXO3a signaling pathways.

Sarcopenia is closely associated with gut dysbiosis. Probiotics alleviate gut dysbiosis. Therefore, we selected probiotics Lactobacillus paracasei P62 (Lp) and Bifidobacterium bifidum P61 (Bb), which suppressed muscle RING-finger protein-1 (MuRF1) expression and NF-κB activation in C2C12 cells, and examined their effects on muscle mass loss and dysfunction in aged mice. Oral administration of Lp, Bb, or their mix (LB) increased grip strength and treadmill running distance and time. They significantly increased muscle weight in aged mice. They also increased AKT activation, PGC1α, SIRT1, and myosin heavy chain (MyHC) expression, MyHC-positive cell population, and cell size in the gastrocnemius (GA) muscle, while FOXO3a and NF-κB activation, MuRF1, muscle atrophy F-box, and p16 expression, and NF-κB+CD11c+ cell population decreased. Furthermore, they reduced cognitive impairment-like behavior, IL-6 expression, FOXO3a activation, and NF-κB-positive cell population in the hippocampus, GA, and colon, while hippocampal brain-derived neurotropic factor expression increased. They shifted gut microbiota composition in aged mice: they increased Akkermansiaceae and Bacteroidaceae populations, which were positively correlated with total muscle weight and MyHC expression, and decreased Odoribacteraceae and Deferribacteriaceae populations, which were positively correlated with MuRF1 and IL-6 expression. LB alleviated sarcopenia- and cognitive impairment-like symptoms more potently than Lp or Bb alone. Based on these findings, probiotics, particularly Lp, Bb, and LB, can alleviate aging-dependent sarcopenia and cognitive impairment by regulating gut microbiota-mediated AKT, NF-κB, and/or FOXO3a signaling pathways.

Alleviation of Cognitive Impairment-like Behaviors, Neuroinflammation, Colitis, and Gut Dysbiosis in 5xFAD Transgenic and Aged Mice by Lactobacillus mucosae and Bifidobacterium longum.

Neuropsychiatric disorders including Alzheimer's disease (AD) may cause gut inflammation and dysbiosis. Gut inflammation-suppressing probiotics alleviate neuropsychiatric disorders. Herein, to understand whether anti-inflammatory probiotics Lactobacillus mucosae NK41 and Bifidobacterium longum NK46, which suppressed tumor necrosis factor (TNF)-α expression in lipopolysaccharide (LPS)-stimulated macrophages, could alleviate cognitive impairment, we first examined their effects on cognitive function, gut inflammation, and gut microbiota composition in 5xFAD-transgenic mice. Oral administration of NK41 or NK46 decreased cognitive impairment-like behaviors, hippocampal amyloid-ß (Aß), TNF-α and interleukin (IL)-1ß expression, hippocampal NF-κB+Iba1+ cell population, and Aß accumulation, while hippocampal brain-derived neurotropic factor (BDNF) and IL-10 expression and BDNF+NeuN+ cell population increased. They also decreased TNF-α and IL-1ß expression and NF-κB+CD11c+ cell population in the colon. They also reduced fecal and blood LPS levels and gut Proteobacteria and Verrucomicrobia populations (including Akkkermansiaceae), which are positively associated with hippocampal TNF-α and fecal LPS levels and negatively correlated with hippocampal BDNF level. However, they increased Odoribactericeae, which positively correlated with BDNF expression level and TNF-α to IL-10 expression ratio. The combination of NK41 and NK46 (4:1, NKc), which potently inhibited TNF-α expression in LPS-stimulated macrophages, additively alleviated cognitive impairment-like behaviors in 5xFAD-transgenic or aged mice. NKc increased hippocampal BDNF+NeuN+ cell population and BDNF expression in 5xFAD-transgenic or aged mice, while hippocampal TNF-α and IL-1ß expression decreased. NKc also decreased TNF-α and IL-1ß expression in the colon and LPS levels in the blood and feces. These findings suggest that gut bacteria and its product LPS may be closely connected with occurrence of cognitive impairment and neuroinflammation and the combination of NK41 and NK46 can additively alleviate cognitive impairment and neuroinflammation by inducing NF-κB-suppressed BDNF expression and suppressing LPS-producing gut bacteria.

Lactobacillus casei and Its Supplement Alleviate Stress-Induced Depression and Anxiety in Mice by the Regulation of BDNF Expression and NF-κB Activation.

Stress-induced depression and anxiety (DA) are closely connected to gastrointestinal inflammation and dysbiosis, which can suppress brain-derived neurotrophic factor (BDNF) in the brain. Herein, we isolated the BDNF expression-inducing probiotics Lactobacillus casei HY2782 and Bifidobacterium lactis HY8002 in lipopolysaccharide-stimulated SH-SY5Y cells. Then, we investigated the effects of HY2782, HY8002, anti-inflammatory L-theanine, and their supplement (PfS, probiotics-fermented L-theanine-containing supplement) on DA in mice exposed to restraint stress (RS) or the fecal microbiota of patients with inflammatory bowel disease and depression (FMd). Oral administration of HY2782, HY8002, or L-theanine alleviated RS-induced DA-like behaviors. They also decreased RS-induced hippocampal interleukin (IL)-1ß and IL-6 levels, as well as NF-κB-positive cell numbers, blood corticosterone level, and colonic IL-1ß and IL-6 levels and NF-κB-positive cell numbers. L-theanine more potently suppressed DA-like behaviors and inflammation-related marker levels than probiotics. However, these probiotics more potently increased RS-suppressed hippocampal BDNF level and BDNF+NeuN+ cell numbers than L-theanine. Furthermore, HY2782 and HY8002 suppressed RS-increased Proteobacteria and Verrucomicrobia populations in gut microbiota. In particular, they increased Lachnospiraceae and Lactobacillacease populations, which are closely positively associated with hippocampal BDNF expression, and suppressed Sutterellaceae, Helicobacteriaceae, Akkermansiaceae, and Enterobacteriaceae populations, which are closely positively associated with hippocampal IL-1ß expression. HY2782 and HY8002 potently alleviated FMd-induced DA-like behaviors and increased FMd-suppressed BDNF, serotonin levels, and BDNF-positive neuronal cell numbers in the brain. They alleviated blood corticosterone level and colonic IL-1ß α and IL-6 levels. However, L-theanine weakly, but not significantly, alleviated FMd-induced DA-like behaviors and gut inflammation. BDNF expression-inducing probiotic (HY2782, HY8002, Streptococcus thermophilus, and Lactobacillus acidophilus)-fermented and anti-inflammatory L-theanine-containing supplement PfS alleviated DA-like behaviors, inflammation-related biomarker levels, and gut dysbiosis more than probiotics or L-theanine. Based on these findings, a combination of BDNF expression-inducing probiotics with anti-inflammatory L-theanine may additively or synergistically alleviate DA and gut dysbiosis by regulating gut microbiota-mediated inflammation and BDNF expression, thereby being beneficial for DA.

Alleviation of Porphyromonas gingivalis or Its Extracellular Vesicles Provoked Periodontitis and Cognitive Impairment by Lactobacillus pentosus NK357 and Bifidobacterium bifidum NK391.

Porphyromonas gingivalis (PG) is closely involved in the outbreak of periodontitis and cognitive impairment (CI). Herein, we examined the effects of anti-inflammatory Lactobacillus pentosus NK357 and Bifidobacterium bifidum NK391 on PG- or its extracellular vesicles (pEVs)-induced periodontitis and CI in mice. Oral administration of NK357 or NK391 significantly decreased PG-induced tumor necrosis factor (TNF)-α, receptor activator of nuclear factors κB (RANK), and RANK ligand (RANKL) expression, gingipain (GP)+lipopolysaccharide (LPS)+ and NF-κB+CD11c+ populations, and PG 16S rDNA level in the periodontal tissue. Their treatments also suppressed PG-induced CI -like behaviors, TNF-α expression and NF-κB-positive immune cells in the hippocampus and colon, while PG-suppressed hippocampal BDNF and N-methyl-D-aspartate receptor (NMDAR) expression increased. The combination of NK357 and NK391 additively alleviated PG- or pEVs-induced periodontitis, neuroinflammation, CI-like behaviors, colitis, and gut microbiota dysbiosis and increased PG- or pEVs-suppressed BDNF and NMDAR expression in the hippocampus. In conclusion, NK357 and NK391 may alleviate periodontitis and dementia by regulating NF-κB, RANKL/RANK, and BDNF-NMDAR signaling and gut microbiota.

Lactobacillus gasseri NK109 and Its Supplement Alleviate Cognitive Impairment in Mice by Modulating NF-κB Activation, BDNF Expression, and Gut Microbiota Composition.

Aging-related gut microbiota dysbiosis initiates gut inflammation and microbiota dysbiosis, which induce the occurrence of psychiatric disorders including dementia. The alleviation of gut microbiota dysbiosis by probiotics is suggested to be able to alleviate psychiatric disorders including cognitive impairment (CI). Therefore, to understand how probiotics could alleviate CI, we examined the effects of anti-inflammatory Lactobacillus gasseri NK109 and its supplement (NS, mixture of NK109 and soybean embryo ethanol extract) on cognitive function in aged (Ag), 5XFAD transgenic (Tg), or mildly cognition-impaired adult fecal microbiota (MCF)-transplanted mice. Oral administration of NK109 or NS decreased CI-like behaviors in Ag mice. Their treatments suppressed TNF-α and p16 expression and NF-κB-activated cell populations in the hippocampus and colon, while BDNF expression was induced. Moreover, they partially shifted the ß-diversity of gut microbiota in Ag mice to those of young mice: they decreased Bifidobacteriaceae, Lactobacillaceae, and Helicobacteriaceae populations and increased Rikenellaceae and Prevotellaceae populations. Oral administration of NK109 or NS also reduced CI-like behaviors in Tg mice. Their treatments induced BDNF expression in the hippocampus, decreased hippocampal TNF-α and Aß expression and hippocampal and colonic NF-κB-activated cell populations. NK109 and NS partially shifted the ß-diversity of gut microbiota in Tg mice: they decreased Muribaculaceae and Rhodospiraceae populations and increased Helicobacteriaceae population. Oral administration of NK109 or NS decreased MCF transplantation-induced CI-like behaviors in mice. NK109 and NS increased hippocampal BDNF expression, while hippocampal and colonic TNF-α expression and NF-κB-activated cell populations decreased. These findings suggest that dementia can fluctuate the gut microbiota composition and NK109 and its supplement NS can alleviate CI with systemic inflammation by inducing BDNF expression and suppressing NF-κB activation and gut microbiota dysbiosis.

Extracellular vesicles derived from Porphyromonas gingivalis induce trigeminal nerve-mediated cognitive impairment.

INTRODUCTION: Porphyromonas gingivalis (PG)-infected periodontitis is in close connection with the development of Alzheimer's disease (AD). PG-derived extracellular vesicles (pEVs) contain inflammation-inducing virulence factors, including gingipains (GPs) and lipopolysaccharide (LPS). OBJECTIVES: To understand how PG could cause cognitive decline, we investigated the effects of PG and pEVs on the etiology of periodontitis and cognitive impairment in mice. METHODS: Cognitive behaviors were measured in the Y-maze and novel object recognition tasks. Biomarkers were measured using ELISA, qPCR, immunofluorescence assay, and pyrosequencing. RESULTS: pEVs contained neurotoxic GPs and inflammation-inducible fimbria protein and LPS. Gingivally exposed, but not orally gavaged, PG or pEVs caused periodontitis and induced memory impairment-like behaviors. Gingival exposure to PG or pEVs increased TNF-α expression in the periodontal and hippocampus tissues. They also increased hippocampal GP+Iba1+, LPS+Iba1+, and NF-κB+Iba1+ cell numbers. Gingivally exposed PG or pEVs decreased BDNF, claudin-5, and N-methyl-D-aspartate receptor expression and BDNF+NeuN+ cell number. Gingivally exposed fluorescein-5-isothiocyanate-labeled pEVs (F-pEVs) were detected in the trigeminal ganglia and hippocampus. However, right trigeminal neurectomy inhibited the translocation of gingivally injected F-EVs into the right trigeminal ganglia. Gingivally exposed PG or pEVs increased blood LPS and TNF-α levels. Furthermore, they caused colitis and gut dysbiosis. CONCLUSION: Gingivally infected PG, particularly pEVs, may cause cognitive decline with periodontitis. PG products pEVs and LPS may be translocated into the brain through the trigeminal nerve and periodontal blood pathways, respectively, resulting in the cognitive decline, which may cause colitis and gut dysbiosis. Therefore, pEVs may be a remarkable risk factor for dementia.

Pharmacokinetic Profiling of Ginsenosides, Rb1, Rd, and Rg3, in Mice with Antibiotic-Induced Gut Microbiota Alterations: Implications for Variability in the Therapeutic Efficacy of Red Ginseng Extracts.

Ginsenoside Rg3 is reported to contribute to the traditionally known diverse effects of red ginseng extracts. Significant individual variations in the therapeutic efficacy of red ginseng extracts have been reported. This study aimed to investigate the effect of amoxicillin on the pharmacokinetics of ginsenosides Rb1, Rd, and Rg3 in mice following the oral administration of red ginseng extracts. We examined the α-diversity and ß-diversity of gut microbiota and conducted pharmacokinetic studies to measure systemic exposure to ginsenoside Rg3. We also analyzed the microbiome abundance and microbial metabolic activity involved in the biotransformation of ginsenoside Rb1. Amoxicillin treatment reduced both the α-diversity and ß-diversity of the gut microbiota and decreased systemic exposure to ginsenoside Rg3 in mice. The area under the curve (AUC) values for Rg3 in control and amoxicillin-treated groups were 247.7 ± 96.6 ng·h/mL and 139.2 ± 32.9 ng·h/mL, respectively. The microbiome abundance and microbial metabolic activity involved in the biotransformation of ginsenoside Rb1 were also altered by amoxicillin treatment. The metabolizing activity was reduced from 0.13 to 0.05 pmol/min/mg on average. Our findings indicate that amoxicillin treatment potentially reduces the gut-microbiota-mediated metabolism of ginsenoside Rg3 in mice given red ginseng extracts, altering its pharmacokinetics. Gut microbiome variations may thus influence individual ginsenoside pharmacokinetics, impacting red ginseng extract's efficacy. Our results suggest that modulating the microbiome could enhance the efficacy of red ginseng.

Chaihu-Shugan-San (Shihosogansan) alleviates restraint stress-generated anxiety and depression in mice by regulating NF-κB-mediated BDNF expression through the modulation of gut microbiota.

BACKGROUND: Chaihu-Shugan-San (CSS, named Shihosogansan in Korean), a Chinese traditional medicine, is frequently used to treat anxiety and depression. Psychiatric disorders including depression are associated with gut dysbiosis. Therefore, to comprehend gut microbiota-involved anti-depressive effect of CSS, we examined its effect on restraint stress (RS)-induced depression and gut dysbiosis in mice METHODS: CSS was extracted with water in boiling water bath and freeze-dried. Anxiety and depression was induced in C57BL/6 mice by exposure to RS. Anxiety- and depression-like behaviors were measured in the light/dark transition and elevated plus maze tasks, forced swimming test, and tail suspension test. Biomarkers were assayed by using the enzyme-linked immunosorbent assay and immunoblotting. The gut microbiota composition was analyzed by Illumina iSeq sequencer. RESULTS: CSS significantly reduced the RS-induced anxiety- and depression-like behaviors in mice. CSS suppressed the RS-induced activation of NF-κB and expression of interleukin (IL)-6 and increased the RS-suppressed expression of brain-derived neurotrophic factor (BDNF). Furthermore, CSS suppressed the RS-induced IL-6 and corticosterone level in the blood and IL-6 expression and myeloperoxidase activity in the colon. CSS decreased the RS-induced γ-Proteobacteria population in gut microbiota, while the RS-suppressed Lactobacillaceae, Prevotellaceae, and AC160630_f populations increased. Fecal transplantation of vehicle-treated control or RS/CSS-treated mice into RS-exposed mice significantly mitigated RS-induced anxity- and depression-like behaviors, suppressed the NF-κB activation in the hippocampus and colon, and reduced the IL-6 and corticosterone levels in the blood. These fecal microbiota transplantations suppressed RS-induced Desulfovibrionaceae and γ-Proteobacteria populations and increased RS-suppressed Lactobacillaceae and Prevotellaceae poulation in the gut microbiota. CONCLUSIONS: CSS alleviated anxiety and depression by inducing NF-κB-involved BDNF expression through the regulation of gut inflammation and microbiota.

Ochratoxin A suppresses proliferation of Sertoli and Leydig cells in mice.

Ochratoxin A (OTA) is a mycotoxin originating from Penicillium and Aspergillus. In addition to toxic effects in various tissues and cells, including neurons, immune cells, hepatocytes, and nephrons, it also causes carcinogenesis and teratogenesis. Although the negative effects of OTA with respect to the pathogenesis of diseases and the malfunction of various organs have been studied widely, the biological signaling mechanisms in testicular cells are less well known. Therefore, we determined the hazardous effect of OTA in two types of testicular cells: TM3 (mouse Leydig cells) and TM4 (mouse Sertoli cells). Treatment with OTA led to a significant decrease in the proliferation of both cell lines, as revealed by an increased proportion of cells in the sub-G1 phase. In addition, the phosphorylation of signaling molecules belonging to the PI3K (Akt, P70S6K, and S6) and MAPK (ERK1/2 and JNK) pathways was regulated by OTA in a dose-dependent manner in TM3 and TM4 cells. Furthermore, the combination treatment of OTA and signaling inhibitors (LY294002, U0126, or SP600125) exerted synergistic antiproliferative effects in TM3 and TM4 cells. OTA also reduced the concentration of calcium ions in the cytosol and mitochondria, which disrupted the calcium homeostasis necessary for maintaining the normal physiological functions of testicular cells. In conclusion, the results of the present study demonstrate the mechanism underlying the antiproliferative effects of OTA in mouse testicular cells. Exposure to OTA may result in abnormal sperm maturation and the failure of spermatogenesis, which leads to male infertility.

Systematic approach identifies RHOA as a potential biomarker therapeutic target for Asian gastric cancer.

Gastric cancer (GC) is a highly heterogeneous disease, in dire need of specific, biomarker-driven cancer therapies. While the accumulation of cancer "Big Data" has propelled the search for novel molecular targets for GC, its specific subpathway and cellular functions vary from patient to patient. In particular, mutations in the small GTPase gene RHOA have been identified in recent genome-wide sequencing of GC tumors. Moreover, protein overexpression of RHOA was reported in Chinese populations, while RHOA mutations were found in Caucasian GC tumors. To develop evidence-based precision medicine for heterogeneous cancers, we established a systematic approach to integrate transcriptomic and genomic data. Predicted signaling subpathways were then laboratory-validated both in vitro and in vivo, resulting in the identification of new candidate therapeutic targets. Here, we show: i) differences in RHOA expression patterns, and its pathway activity, between Asian and Caucasian GC tumors; ii) in vitro and in vivo perturbed RHOA expression inhibits GC cell growth in high RHOA-expressing cell lines; iii) inverse correlation between RHOA and RHOB expression; and iv) an innovative small molecule design strategy for RHOA inhibitors. In summary, RHOA, and its oncogenic signaling pathway, represent a strong biomarker-driven therapeutic target for Asian GC. This comprehensive strategy represents a promising approach for the development of "hit" compounds.

Improving gastric cancer preclinical studies using diverse in vitro and in vivo model systems.

BACKGROUND: "Biomarker-driven targeted therapy," the practice of tailoring patients' treatment to the expression/activity levels of disease-specific genes/proteins, remains challenging. For example, while the anti-ERBB2 monoclonal antibody, trastuzumab, was first developed using well-characterized, diverse in vitro breast cancer models (and is now a standard adjuvant therapy for ERBB2-positive breast cancer patients), trastuzumab approval for ERBB2-positive gastric cancer was largely based on preclinical studies of a single cell line, NCI-N87. Ensuing clinical trials revealed only modest patient efficacy, and many ERBB2-positive gastric cancer (GC) patients failed to respond at all (i.e., were inherently recalcitrant), or succumbed to acquired resistance. METHOD: To assess mechanisms underlying GC insensitivity to ERBB2 therapies, we established a diverse panel of GC cells, differing in ERBB2 expression levels, for comprehensive in vitro and in vivo characterization. For higher throughput assays of ERBB2 DNA and protein levels, we compared the concordance of various laboratory quantification methods, including those of in vitro and in vivo genetic anomalies (FISH and SISH) and xenograft protein expression (Western blot vs. IHC), of both cell and xenograft (tissue-sectioned) microarrays. RESULTS: The biomarker assessment methods strongly agreed, as did correlation between RNA and protein expression. However, although ERBB2 genomic anomalies showed good in vitro vs. in vivo correlation, we observed striking differences in protein expression between cultured cells and mouse xenografts (even within the same GC cell type). Via our unique pathway analysis, we delineated a signaling network, in addition to specific pathways/biological processes, emanating from the ERBB2 signaling cascade, as a potential useful target of clinical treatment. Integrated analysis of public data from gastric tumors revealed frequent (10 - 20 %) amplification of the genes NFKBIE, PTK2, and PIK3CA, each of which resides in an ERBB2-derived subpathway network. CONCLUSION: Our comprehensive bioinformatics analyses of highly heterogeneous cancer cells, combined with tumor "omics" profiles, can optimally characterize the expression patterns and activity of specific tumor biomarkers. Subsequent in vitro and in vivo validation, of specific disease biomarkers (using multiple methodologies), can improve prediction of patient stratification according to drug response or nonresponse.

HNF4α is a therapeutic target that links AMPK to WNT signalling in early-stage gastric cancer.

BACKGROUND: Worldwide, gastric cancer (GC) is the fourth most common malignancy and the most common cancer in East Asia. Development of targeted therapies for this disease has focused on a few known oncogenes but has had limited effects. OBJECTIVE: To determine oncogenic mechanisms and novel therapeutic targets specific for GC by identifying commonly dysregulated genes from the tumours of both Asian-Pacific and Caucasian patients. METHODS: We generated transcriptomic profiles of 22 Caucasian GC tumours and their matched non-cancerous samples and performed an integrative analysis across different GC gene expression datasets. We examined the inhibition of commonly overexpressed oncogenes and their constituent signalling pathways by RNAi and/or pharmacological inhibition. RESULTS: Hepatocyte nuclear factor-4α (HNF4α) upregulation was a key signalling event in gastric tumours from both Caucasian and Asian patients, and HNF4α antagonism was antineoplastic. Perturbation experiments in GC tumour cell lines and xenograft models further demonstrated that HNF4α is downregulated by AMPKα signalling and the AMPK agonist metformin; blockade of HNF4α activity resulted in cyclin downregulation, cell cycle arrest and tumour growth inhibition. HNF4α also regulated WNT signalling through its target gene WNT5A, a potential prognostic marker of diffuse type gastric tumours. CONCLUSIONS: Our results indicate that HNF4α is a targetable oncoprotein in GC, is regulated by AMPK signalling through AMPKα and resides upstream of WNT signalling. HNF4α may regulate 'metabolic switch' characteristic of a general malignant phenotype and its target WNT5A has potential prognostic values. The AMPKα-HNF4α-WNT5A signalling cascade represents a potentially targetable pathway for drug development.

A pathway-based approach for identifying biomarkers of tumor progression to trastuzumab-resistant breast cancer.

Although trastuzumab is a successful targeted therapy for breast cancer patients with tumors expressing HER2 (ERBB2), many patients eventually progress to drug resistance. Here, we identified subpathways differentially expressed between trastuzumab-resistant vs. -sensitive breast cancer cells, in conjunction with additional transcriptomic preclinical and clinical gene datasets, to rigorously identify overexpressed, resistance-associated genes. From this approach, we identified 32 genes reproducibly upregulated in trastuzumab resistance. 25 genes were upregulated in drug-resistant JIMT-1 cells, which also downregulated HER2 protein by >80% in the presence of trastuzumab. 24 genes were downregulated in trastuzumab-sensitive SKBR3 cells. Trastuzumab sensitivity was restored by siRNA knockdown of these genes in the resistant cells, and overexpression of 5 of the 25 genes was found in at least one of five refractory HER2 + breast cancer. In summary, our rigorous computational approach, followed by experimental validation, significantly implicate ATF4, CHEK2, ENAH, ICOSLG, and RAD51 as potential biomarkers of trastuzumab resistance. These results provide further proof-of-concept of our methodology for successfully identifying potential biomarkers and druggable signal pathways involved in tumor progression to drug resistance.