Obesity affects brain cortex gene expression in an APOE genotype and sex dependent manner.

OBJECTIVE: Obesity is the top modifiable risk factor for Alzheimer's disease. We hypothesized that high fat diet (HFD)-induced obesity alters brain transcriptomics in APOE-genotype and sex dependent manners. Here, we investigated interactions between HFD, APOE, and sex, using a knock-in mouse model of the human APOE3 and APOE4 alleles. METHODS: Six-month-old APOE3-TR and APOE4-TR mice were treated with either HFD or control chow. After 4 months, total RNA was extracted from the cerebral cortices and analyzed by poly-A enriched RNA sequencing on the Illumina platform. RESULTS: Female mice demonstrated profound HFD-induced transcriptomic changes while there was little to no effect in males. In females, APOE3 brains demonstrated about five times more HFD-induced transcriptomic changes (399 up-regulated and 107 down-regulated genes) compared to APOE4 brains (30 up-regulated and 60 down-regulated). Unsupervised clustering analysis revealed two gene sets that responded to HFD in APOE3 mice but not in APOE4 mice. Pathway analysis demonstrated that HFD in APOE3 mice affected cortical pathways related to feeding behavior, blood circulation, circadian rhythms, extracellular matrix, and cell adhesion. CONCLUSIONS: Female mice and APOE3 mice have the strongest cortical transcriptomic responses to HFD related to feeding behavior and extracellular matrix remodeling. The relative lack of response of the APOE4 brain to stress associated with obesity may leave it more susceptible to additional stresses that occur with aging and in AD.

Electrochemotherapy with Irreversible Electroporation and FOLFIRINOX Improves Survival in Murine Models of Pancreatic Adenocarcinoma.

BACKGROUND: Previously published work has demonstrated that combining gemcitabine with irreversible electroporation (IRE) results in increased drug delivery to pancreatic adenocarcinoma cells in vivo. This study assessed the efficacy of IRE + gemcitabine and IRE + FOLFIRINOX (5-fluorouracil, leucovorin, irinotecan, and oxaliplatin), the impact of the superior regimen on survival, and the safety of electrochemotherapy in human subjects. METHODS: Histologic analysis was performed after in vitro and in vivo treatment of S2013 and Panc-1 pancreatic cancer cells and S2013 orthotopic tumors, respectively, and levels of apoptotic machinery and cell cycle proteins were evaluated using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and Western blot. RESULTS: Electrochemotherapy (ECT) with IRE and FOLFIRINOX resulted in increased tumor cells apoptosis compared with gemcitabine, gemcitabine + IRE, and FOLFIRINOX alone, and significantly improved overall survival when compared with mice treated with IRE or FOLFIRINOX. Increased tumor cell apoptosis, caspase-3 mRNA, active caspase-3 protein, and decreased cell proliferation were noted at the time of death or euthanasia in the ECT group compared with folinic acid alone. In five patients, ECT with either FOLFIRINOX or gemcitabine was well-tolerated and resulted in no dose-limiting toxicities. CONCLUSIONS: ECT thus results in synergistic antitumor activity compared with either treatment modality used alone, resulting in increased tumor cell apoptosis as well as decreased tumor cell proliferation and improved overall survival. Pilot data suggest that ECT represents a promising modality for the treatment of patients with locally advanced pancreatic cancer. TRIAL REGISTRATION: The human subject portion of this work was conducted as part of an investigator-initiated clinical trial at the University of Louisville (NCT03484299).

Epigenetic modulation enhances immunotherapy for hepatocellular carcinoma.

BACKGROUND: Anti-PDL-1 immunotherapy for Hepatocellular Carcinoma (HCC) demonstrated a mixed response. Polycomb Repressor Complex 2(PRC2) contributes to the initiation and progression of HCC by suppressing tumor antigens and inhibiting an immune response. Two components of epigenetic modulation are Enhancer of Zeste Homolog 2 (EZH2, the catalytic component of PRC2) and DNA Methyltransferase 1 (DNMT1). We aim to investigate the potential role of epigenetic therapy targeting EZH2 and DNMT1 as a novel strategy to modulate immunotherapy response in HCC. METHODS: HepG2, Hep3B, and Hepa1-6 HCC cell lines were treated with EZH2 inhibitor (DZNep) and DNMT1 inhibitor (5-Azacytidine) with and without anti-PDL-1. Quantitative RT-PCR and immunohistochemistry were performed to evaluate the expression of tumor suppressors, tumor antigens, and Th1 chemokines. In-vivo C57/LJ immunocompetent mice model with subcutaneous tumor inoculation was performed with intraperitoneal drug injections. RESULTS: There was a significant upregulation of Th1 chemokines in HepG2 (CXCL9 5.5 ±â€¯0.2 relative fold change; CXCL10 1.44 × 103 ±â€¯37 relative fold change) and Hep3B (CXCL 9 6.85 × 103 ±â€¯1.3 × 103 relative fold change; CXCL 10 2.15 × 103 ±â€¯3.1 × 102 relative fold change). Additionally, there was a significant induction of cancer testis antigens NY-ESO-1 (3.6-3.7 ±â€¯0.3 relative fold change) and LAGE (8.3-11.7 ±â€¯1.9 relative fold change). In vivo model demonstrated statistically significant tumor regression in the combination treatment group (0.02 g ±â€¯0.02) compared to epigenetic therapy (0.63 g ±â€¯0.61) or immunotherapy alone (0.15 g ±â€¯0.21) with untreated control (2.4 g ±â€¯0.71). There was significantly increased trafficking of cytotoxic T- lymphocytes and associated apoptosis for the combination treatment group compared to epigenetic or immunotherapy alone. CONCLUSIONS: This study demonstrates that epigenetic modulation could be a novel potential strategy to augment immunotherapy for HCC by stimulating T cell trafficking into tumor microenvironment via activation of transcriptionally repressed chemokine genes responsible for T-cell trafficking, inducing previously silent neoantigens for immune targets, and allowing tumor regression as a result. A clinical trial of this feasible combination therapy of these clinically available agents is warranted.

Evaluating the Regulatory Immunomodulation Effect of Irreversible Electroporation (IRE) in Pancreatic Adenocarcinoma.

BACKGROUND: Irreversible electroporation (IRE) has been demonstrated as an effective local method for locally advanced (stage 3) pancreatic adenocarcinoma. Immune regulatory T cells (Tregs) induce immunosuppression of tumors by inhibiting patients' anti-tumor adaptive immune response. This study aimed to evaluate the immunomodulation effect of IRE to identify an ideal time point for potential adjuvant immunotherapy. METHODS: This study prospectively evaluated an institutional review board-approved study of patients undergoing either in situ IRE or pancreatectomy. Patient blood samples were collected at different time points (before surgery [preOP] and on postoperative day [POD] 1, POD3, and POD5). Peripheral blood mononuclear cells (PBMCs) were isolated and evaluated for three different CD4 + Treg subsets (CD25 + CD4 +, CD4 + CD25 + FoxP3 +, CD4 + CD25 + FoxP3 -) by flow cytometry and analyzed for median fold change (MFC) between each two consecutive time points (MFC = log2(T2/T1)). RESULTS: The study analyzed 15 patients with in situ IRE (n = 11) or pancreatectomy (PAN) (n = 4). In both groups, CD25 + CD4 + Tregs decreased on POD1 followed by a steady increase in pancreatectomy, whereas the trend in the IRE group reversed between D3 and D5 (MFC: IRE [- 0.01], PAN [+ 0.39]). For each period, CD4 + CD25 + FoxP3 + Tregs showed the most dramatic inverse effect, with D3 to D5 showing the most change (MFC: IRE [- 0.18], PAN [+ 0.39]). Also, CD4 + CD25 + FoxP3 - Tregs showed an inverse effect between D3 and D5 (MFC: IRE [- 0.25], PAN [+ 0.49]). Altogether, the Treg trend was inversely affected by the in situ IRE procedure, with the greatest cumulative significant change for all three Treg subsets between D3 and D5 (MFC ± SEM: IRE [- 0.24 ± 0.05], PAN [+ 0.37 ± 0.02]; p = 0.016). CONCLUSIONS: The study data suggest that in situ IRE procedure-mediated Treg attenuation between POD3 and POD5 can provide a clinical window of opportunity for potentiating clinical efficacy in combination with immunotherapy.

Enrichment of cancer stem cells via ß-catenin contributing to the tumorigenesis of hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is among the deadliest cancers due to its heterogeneity, contributing to chemoresistance and recurrence. Cancer stem-like cells (CSCs) are suggested to play an important role in HCC tumorigenesis. This study investigates the role of Wnt/ß-catenin pathway in CSC enrichment and the capabilities of these CSCs in tumor initiation in orthotopic immunocompetent mouse model. METHODS: HCC-CSCs were enriched using established serum-free culture method. Wnt/ß-catenin pathway activation and its components were analyzed by western blot and qRT-PCR. The role of ß-catenin in enrichment of CSC spheroids was confirmed using siRNA interference. Tumorigenic capabilities were confirmed using orthotopic immunocompetent mouse model by injecting 2 × 106 Hepa1-6 CSC spheroids or control cells in upper left liver lobe. RESULTS: The serum-free cultured Hepa1-6 cells demonstrated self-renewal, spheroid formation, higher EpCAM expression, increased Hoechst-33342 efflux, and upregulated Wnt/ß-catenin signaling. Wnt/ß-catenin pathway upregulation was implicated with the downstream targets, i.e., c-MYC, Cyclin-D1, and LEF1. Also, we found that GSK-3ß serine-9 phosphorylation increased in Hepa1-6 spheroids. Silencing ß-catenin by siRNA reversed spheroid formation phenotype. Mice injected with Hepa1-6 CSC spheroids showed aggressive tumor initiation and growth compared with mice injected with control cells. CONCLUSIONS: Successfully induced Hepa1-6 spheroids were identified with CSC-like properties. Aberrant ß-catenin upregulation mediated by GSK-3ß was observed in the Hepa1-6 spheroids. The ß-catenin mediated CSC enrichment in the induced spheroids possesses the capability of tumor initiation in immunocompetent mice. Our study suggests plausible cell dedifferentiation mediated by ß-catenin contributes to CSC-initiated HCC tumor growth in vivo.

Exposure to bile acids alters the intracellular location and function of MnSOD in Barrett's esophagus.

BACKGROUND: Oxidative stress secondary to bile-acid exposure has been associated with metaplastic degeneration of normal esophageal mucosa into Barrett's esophagus (BE) cells and eventually esophageal adenocarcinoma. We previously reported that the macromolecular response of BE cells to this stress was largely regulated by the expression of manganese-dependent mitochondrial superoxide dismutase (MnSOD). As the mitochondrion plays a vital role in MnSOD activation, this study sought to determine the location and activity of MnSOD within BE cells after exposure to oxidative stress. METHODS: A human BE cell line, BAR-T cell, was exposed 0.4 mM concentrations of taurocholic acid (Tau) or a 0.4 mM 1:1 mixture of bile salts for 4 h. Cell viability was performed with 3-(4, 5-dimthyl-thiazol-2-yl)-2, 5-diphenyltetrazolium bromide assays. Proteins were extracted and separated into mitochondrial, nuclear, and cytoplasmic fractions followed by analysis by a western blot and enzymatic activities. RESULTS: BAR-T cell showed resistance to the bile-salt insults. Expression of MnSOD was significantly increased in the cells exposed to a mixture of bile acids and Tau versus control. Mitochondria MnSOD is abundant and highly active. Nuclear fraction displayed presence of both MnSOD and Cu/zinc superoxide dismutase secondary to bile-acid exposure; however, the MnSOD was inactive in nuclear fraction. CONCLUSIONS: This is the first study to specifically evaluate cellular fraction MnSOD expression, increased in BE cells in response to the oxidative stress of bile exposure. Mitochondrial MnSOD contributes to resistance of BAR-T cells to the bile-salt insults. Further investigation is required to determine the potential correlation between bile exposure and BE to adenocarcinoma progression via MnSOD-mediated cell signaling.

Evaluation of Probe Angles for Synchronous Waveform 915-MHz Microwave Ablation.

BACKGROUND: The purpose of this study is to compare the ablation performance between a synchronous microwave ablation (MWA) system and a commercially available asynchronous system in ex vivo bovine liver and evaluate the efficacy of ablation at varying entrance angles. MATERIALS AND METHODS: Two 915-MHz MWA systems were used in bench top ex vivo bovine livers with various conditions (synchronous versus asynchronous). Using synchronous technology ablations to liver, kidney, or lung at angles of 0, 15, 30, and 90° were evaluated. RESULTS: Synchronous and asynchronous MWA systems created mean ablation zone volumes of 26.4 and 15.8 cm3, 62.9 and 45.4 cm3, 90.8 and 56.4, and 75.7 and 54.8 cm3 with single, double (2 microwave probes in use simultaneous) (2 cm spacing), and triple (three probes in use simultaneously) (2 cm and 3 cm spacing) antennae configurations, respectively; adjusted P-values ≤ 0.006. Ablation defects were similar across all groups when evaluated for entrance angle. Specifically, when comparing 0-degree angle to all other angles, achieved zones of ablation (ZA) were similar (mean ± standard deviation for 0-degree versus all other angles: 8.72 ± 4.84 versus 9.38 ± 4.11 cm2, P = 0.75). The use of the long-tip probe resulted in a statistically significant increase in the achieved ZA when compared to the short tip probe (10.9 ± 4.3 versus 6.5 ± 2.4, respectively; P = 0.01). CONCLUSIONS: Newly developed synchronous microwave technology creates significantly larger ablation zones when compared to an existing asynchronous commercially available system. The angle of approach does not affect the resulting ZA. This is clinically relevant as true 0-degree angle is often difficult to obtain.

Behavioral and transcriptomic effects of the cancer treatment tamoxifen in mice.

Introduction: Tamoxifen is a common treatment for estrogen receptor-positive breast cancer. While tamoxifen treatment is generally accepted as safe, there are concerns about adverse effects on cognition. Methods: We used a mouse model of chronic tamoxifen exposure to examine the effects of tamoxifen on the brain. Female C57/BL6 mice were exposed to tamoxifen or vehicle control for six weeks; brains of 15 mice were analyzed for tamoxifen levels and transcriptomic changes, and an additional 32 mice were analyzed through a battery of behavioral tests. Results: Tamoxifen and its metabolite 4-OH-tamoxifen were found at higher levels in the brain than in the plasma, demonstrating the facile entry of tamoxifen into the CNS. Behaviorally, tamoxifen-exposed mice showed no impairment in assays related to general health, exploration, motor function, sensorimotor gating, and spatial learning. Tamoxifen-treated mice showed a significantly increased freezing response in a fear conditioning paradigm, but no effects on anxiety measures in the absence of stressors. RNA sequencing analysis of whole hippocampi showed tamoxifen-induced reductions in gene pathways related to microtubule function, synapse regulation, and neurogenesis. Discussion: These findings of the effects of tamoxifen exposure on fear conditioning and on gene expression related to neuronal connectivity suggest that there may be CNS side effects of this common breast cancer treatment.

Extracellular vesicles released after cranial radiation: An insight into an early mechanism of brain injury.

Cranial radiation is important for treating both primary brain tumors and brain metastases. A potential delayed side effect of cranial radiation is neurocognitive function decline. Early detection of CNS injury might prevent further neuronal damage. Extracellular vesicles (EVs) have emerged as a potential diagnostic tool because of their unique membranous characteristics and cargos. We investigated whether EVs can be an early indicator of CNS injury by giving C57BJ/6 mice 10 Gy cranial IR. EVs were isolated from sera to quantify: 1) number of EVs using nanoparticle tracking analysis (NTA); 2) Glial fibrillary acidic protein (GFAP), an astrocyte marker; and 3) protein-bound 4-hydroxy-2-nonenal (HNE) adducts, an oxidative damage marker. Brain tissues were prepared for immunohistochemistry staining and protein immunoblotting. The results demonstrate: 1) increased GFAP levels (p < 0.05) in EVs, but not brain tissue, in the IR group; and 2) increased HNE-bound protein adduction levels (p < 0.05). The results support using EVs as an early indicator of cancer therapy-induced neuronal injury.

Epigenetic modulation enhances immunotherapy for pancreatic ductal adenocarcinoma.

Objectives: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with a poor prognosis. PDAC has poor response to immunotherapy because of its unique tumour microenvironment (TME). In an attempt to stimulate immunologically silent pancreatic cancer, we investigated the role of epigenetic therapy in modulating the TME to improve immunogenicity. Methods: In vitro human PDAC cell lines MiaPaca2 and S2-013 were treated with 5µ m 3-Deazaneplanocin A (DZNep, an EZH2 inhibitor) and 5 µ m 5-Azacytidine (5-AZA, a DNMT1 inhibitor). In vivo orthotopic murine tumour models using both murine PAN02 cells and KPC cells inoculated in immunocompetent C56/BL7 mice were treated with anti-PD-L1 combined with DZNep and 5-AZA. Short hairpin knockdown (KD) of EZH2 and DNMT1 in PAN02 cells for the orthotopic murine tumour model was established to validate the drug treatment (DZNep and 5-AZA). qRT-PCR and microarray assays were performed for the evaluation of Th1-attracting chemokines and cancer-associated antigen induction. Results: Drug treatments induced significant upregulation of gene expressions of Th1-attracting chemokines, CXCL9 and CXCL10, and the cancer-testis antigens, NY-ESO-1, LAGE and SSX-4 (P < 0.05). In orthotopic tumour models, inoculation of PAN02 cells or KPC cells demonstrated significant tumour regression with corresponding increased apoptosis and infiltration of cytotoxic T lymphocytes in the combination treatment group. In the orthotopic Pan02-KD model, the anti-PD-L1 treatment also caused significant tumour regression. Conclusion: We demonstrate that immunotherapy for PDAC can be potentiated with epigenetic therapy by increasing cancer-associated antigen expression and increased T-cell trafficking across the immunosuppressive tumour microenvironment via upregulation of the repressed chemokines and increased apoptosis with subsequent tumour regression.

Carcinogenetic initiation contributed by EpCAM+ cancer cells in orthotopic HCC models of immunocompetent and athymic mice.

PURPOSE: Overexpression of epithelial cell adhesion molecule (EpCAM) correlates with poor prognosis, therapeutic failure and early tumor recurrence in hepatocellular carcinoma (HCC) patients. The tumor microenvironment dictates the fate of tumor-initiating cancer stem cells (CSCs); however, very limited studies were attempted to evaluate CSC tumorigenesis in the liver microenvironment. Here, we have systemically investigated the role of EpCAM+ cancer cells in tumor initiation in orthotopic HCC models. RESULTS: Control mice and the mice with bland steatosis failed to develop tumors. In the mice with steatohepatitis, EpCAM+ CSCs have shown significantly increased ability in terms of tumor initiation and growth, compared to that with EpCAM- non-CSCs inoculation (p < 0.005). For Hep3B inoculation, EpCAM-High group has shown significantly higher tumor growth compared with EpCAM-Low (p < 0.005). For HepG2 inoculation, both EpCAM-High and EpCAM-Low groups confirmed similar tumor incidence and growth. METHODS: Diet-induced compromised microenvironments were established to mimic clinical fatty liver and non-alcoholic steatohepatitis (NASH) patients and the tumorigenic capabilities of Hepa1-6 cells were evaluated. CSCs were enriched by spheroid culture and labeled with copGFP for EpCAM+ CSCs and with mCherry for non-CSCs. FACS-sorted cells were inoculated into left liver lobes, and tumor growth was monitored by high-frequency ultrasound. The subpopulations of Hep3B and HepG2 cells in terms of EpCAM-Low and EpCAM-High were evaluated in the orthotopic model of athymic mice. CONCLUSIONS: NASH microenvironment promotes the EpCAM+ CSCs initiated tumorigenesis in immunocompetent mouse model. Differential EpCAM expression demonstrates distinct tumor biology in athymic mouse models.

Lack of FGF21 promotes NASH-HCC transition via hepatocyte-TLR4-IL-17A signaling.

Rationale: Hepatocellular carcinoma (HCC) has been increasingly recognized in nonalcoholic steatohepatitis (NASH) patients. Fibroblast growth factor 21 (FGF21) is reported to prevent NASH and delay HCC development. In this study, the effects of FGF21 on NASH progression and NASH-HCC transition and the potential mechanism(s) were investigated. Methods: NASH models and NASH-HCC models were established in FGF21Knockout (KO) mice to evaluate NASH-HCC transition. IL-17A signaling was investigated in the isolated hepatic parenchymal cells, splenocytes, and hepatocyte and HCC cell lines. Results: Lack of FGF21 caused significant up-regulation of the hepatocyte-derived IL-17A via Toll-like receptor 4 (TLR4) and NF-κB signaling. Restoration of FGF21 alleviated the high NAFLD activity score (NAS) and attenuated the TLR4-triggered hepatocyte-IL-17A expression. The HCC nodule number and tumor size were significantly alleviated by treatments of anti-IL-17A antibody. Conclusion: This study revealed a novel anti-inflammatory mechanism of FGF21 via inhibiting the hepatocyte-TLR4-IL-17A signaling in NASH-HCC models. The negative feedback loop on the hepatocyte-TLR4-IL-17A axis could be a potential anti-carcinogenetic mechanism for FGF21 to prevent NASH-HCC transition.

Utilizing Contrast-Enhanced Ultrasound Imaging for Evaluating Fatty Liver Disease Progression in Pre-clinical Mouse Models.

We developed a protocol to investigate and optimize the application of contrast-enhanced ultrasound (CEUS) to non-invasive diagnosis of progressing fatty liver disease in mouse models. Eighteen 4-wk-old male C57 L/J mice were randomly assigned to one of the three groups and placed on a control diet, high-fat diet or non-alcoholic steatohepatitis diet for the next 10 wk. After 14 wk, B-mode imaging and CEUS imaging using a VisualSonics Vevo2100 system were performed. CEUS imaging and data analysis using three different parameters-peak enhancement, wash-in rate and wash-in perfusion index-revealed a significant decrease in representative blood flow in the high-fat diet group versus controls and a further significant decrease in the non-alcoholic steatohepatitis group (p < 0.001; n = 6/group). In conclusion, compared with B-mode imaging, non-targeted CEUS imaging was more sensitive in diagnosing early-stage fatty infiltration-mediated vascularity changes in liver parenchyma and provided a more accurate steatohepatitis diagnosis in mouse models.

Serological biomarkers associate ultrasound characteristics of steatohepatitis in mice with liver cancer.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) covers a spectrum of lesions ranging from steatosis to a complex pattern, nonalcoholic steatohepatitis (NASH). Ultrasonography provides important information on hepatic architecture for steatosis. NASH patients have an increased risk of hepatocellular carcinoma (HCC). Early detection of NASH is critical for clinicians to advise on necessary treatments to prevent the onset of HCC. METHODS: We established a NASH-HCC mouse model using diethylnitrosamine as a carcinogen to induce HCC and a high-fat diet to induce metabolic disorders. Characteristics of ultrasound imaging and potential serum biomarkers were investigated for detection of steatohepatitis and HCC in mice. RESULTS: The data suggested that ultrasound imaging of hyperechoic masses was potentially linked to the gross finding of HCC nodules, which was further confirmed by the histology. Positive correlation between serum fibroblast growth factor 15 and acoustic attenuation coefficient was found in mice with steatohepatitis. Combined with the serum markers, the increased acoustic attenuation coefficient could be a useful diagnostic parameter of ultrasound imaging for NASH detection. CONCLUSIONS: This study demonstrates that a combination of serum fibroblast growth factor 15 and acoustic attenuation coefficient could be a sensitive marker for steatohepatitis and to predict carcinogenic initiation and progression of HCC in mice. These results might help for the design of ultrasound and surrogate markers in screening NASH patients who could be at risk of HCC.

Up-regulation of FGF15/19 signaling promotes hepatocellular carcinoma in the background of fatty liver.

BACKGROUND: Upregulated fibroblast growth factor 19 (FGF19) expression in human hepatocellular carcinoma (HCC) specimens is associated with tumor progression and poor prognosis. Nonalcoholic steatohepatitis (NASH) patients are at high risk for malignant transformation into HCC. METHODS: A steatohepatitis-HCC model was established in male C57L/J mice treated with N-nitrosodiethylamine (DEN) and high-fat diet (HFD). A mouse HCC cell line (Hepa1-6) and a mouse hepatocyte line (FL83B) were used to elucidate the mechanism by free fatty acids (FFA) treatment. FGF15, the mouse orthologue of FGF19, and it receptor fibroblast growth factor receptor4 (FGFR4) as well as co-receptor ß-klotho were studied. FGF19 signaling was also studied in human samples of HCC with steatohepatitis. RESULTS: HCC incidence and tumor volume were significantly increased in the DEN+HFD group compared to that in the DEN+control diet (CD) group. Increased levels of FGF15/FGFR4/ß-klotho, aberrant epithelial-mesenchymal transition (EMT) and Wnt/ß-catenin signaling were detected in DEN+HFD mice. Blockage of the FGF15 signal can attenuate cell migration ability and aberrant EMT and Wnt/ß-catenin signaling. CONCLUSIONS: Up-regulated FGF15/FGFR4 signaling promoted the development of HCC by activation of EMT and Wnt/ß-catenin signaling in the lipid metabolic disorder microenvironment. Further investigation of FGF19/FGFR4 signaling is important for potential early diagnosis and therapeutic targeting in HCC patients.

Drug-induced expression of EpCAM contributes to therapy resistance in esophageal adenocarcinoma.

BACKGROUND: With a less than 5% overall survival rate, esophageal adenocarcinoma (EAC) is one of the leading causes of death in the United States. Epithelial cell adhesion molecule (EpCAM) is a cancer stem cell (CSC) marker that is expressed in various epithelial carcinomas, including EAC. Accumulating evidence indicates that CSC subpopulations can initiate cancer development and, in addition, drive metastasis, recurrence and drug resistance. It has also been reported that EpCAM up-regulation in EAC may lead to an aggressive behavior and, thus, an adverse clinical outcome. Here, we aimed to determine whether treatment with standard chemotherapeutic agents may induce EpCAM expression and, concomitantly, increases in malignant potential and drug resistance in EAC. METHODS: EpCAM expression was assessed in 20 primary human EAC/adjacent normal tissues, as well as in a human EAC-derived cell line (OE-19), in a pre-malignant Barrett's Esophagus cell line (Bar-T) and in a benign esophageal cell line (HET 1-A), using immunohistochemistry, Western blotting and qRT-PCR, respectively. Drug-induced resistance was investigated in OE-19-derived spheres treated with (a combination of) adriamycin, cisplatin and 5-fluorouracil (ACF) using survival, adhesion and flow cytometric assays, respectively, and compared to drug resistance induced by standard chemotherapeutic agents (CTA). Finally, ACF treatment-surviving cells were evaluated for their tumor forming capacities both in vitro and in vivo using spheroid formation and xenograft assays, respectively. RESULTS: High EpCAM expression was observed in esophageal cancer tissues and esophageal cancer-derived cell lines, but not in adjacent benign esophageal epithelia and benign esophageal cell lines (HET 1-A and Bar-T). The OE-19 cell spheres were drug resistant and EpCAM expression was significantly induced in the OE-19 cell spheres compared to the non-sphere OE-19 cells. When OE-19 cell spheres were challenged with ACF, the EpCAM mRNA and protein levels were further up-regulated up to 48 h, whereas a decreased EpCAM expression was observed at 72 h. EpCAM down-regulation by RNA interference increased the ACF efficacy to kill OE-19 cells. Increased EpCAM expression coincided with the CSC marker CD90 and was associated with an aggressive growth pattern of OE-19 cell spheres in vivo. CONCLUSIONS: From our data we conclude that an ACF-induced increase in EpCAM expression reflects the selection of a CSC subpopulation that underlies tumor development and drug resistance in EAC.

Up-regulation of fibroblast growth factor 19 and its receptor associates with progression from fatty liver to hepatocellular carcinoma.

BACKGROUND: Human fibroblast growth factor 19 (FGF19), its receptor (FGFR4) and EpCAM play an important role in cell proliferation, differentiation, motility, and overexpression have been linked to hepatocellular carcinoma (HCC). The aim of this study was to evaluate the FGF19 signals responsible for the progression of HCC arising from fatty liver. RESULTS: FGF19 level was significantly increased in the HCC patients' serum compared to non-HCC controls. The IHC results demonstrated significant increases of protein expressions of FGF19, FGFR4 and EpCAM in specimens with fatty liver, NASH, cirrhosis, and HCC compared to healthy liver tissue. There was a significant positive correlation between the protein expressions (FGF19, FGFR4, and EpCAM) and histopathologic changes from FL to HCC. Furthermore, FGF19 was positively correlated with FGFR4 and with EpCAM. MATERIALS AND METHODS: FGF19 protein levels in serum and tissues were determined by ELISA assay. The FGFR4, and EpCAM expression and tissue distribution were further evaluated by immunohistochemical staining in tissue array samples. FGF19, FGFR4 and EpCAM expressions between the different histologic stages of fatty liver steatohepatitis-cirrhosis-HCC carcinogenesis sequence were compared to healthy hepatic tissue. CONCLUSIONS: Overexpression of FGF19/FGFR4 significantly correlated with EpCAM as a marker of hepatic cancer stem cells within the fatty liver-steatosis-cirrhosis-HCC sequence. IMPACT: This is the first study to elucidate FGF19/FGFR4 signaling in favor of HCC cells developing as indicated by increased EpCAM within the carcinogenesis sequence from fatty liver to hepatocellular carcinoma. Our study has the potential to yield novel and cost effective screening strategies for HCC patients.