Clinical utility of postablation liver tumor biopsy and possibility of gene mutation analysis.

AIM: Radiofrequency ablation (RFA) is regarded as a first-line treatment for hepatocellular carcinoma (HCC) at an early stage. When treated with RFA, tumor biopsy may not be performed due to the risk of neoplastic seeding. We previously revealed that the risk of neoplastic seeding is significantly reduced by performing biopsies after RFA. In this study, we investigated the possibility of pathological evaluation and gene mutation analysis of post-RFA tumor specimens. METHODS: Radiofrequency ablation was undertaken on diethylnitrosamine-induced mouse liver tumor, and tumor samples with or without RFA were subjected to whole exome sequencing. Post-RFA human liver tumor specimens were used for detection of TERT promoter mutations and pathological assessment. RESULTS: The average somatic mutation rate, sites of mutation, and small indels and base transition patterns were comparable between the nontreated and post-RFA tumors. We identified 684 sites of nonsynonymous somatic substitutions in the nontreated tumor and 704 sites of nonsynonymous somatic substitutions in the post-RFA tumor, with approximately 85% in common. In the human post-RFA samples, the TERT promoter mutations were successfully detected in 40% of the cases. Pathological evaluation was possible with post-RFA specimens, and in one case, the diagnosis of adenocarcinoma was made. CONCLUSION: Our findings suggest that post-RFA liver tumor biopsy is a useful and safe method for obtaining tumor samples that can be used for gene mutation analysis and for pathological assessment.

HNF1B-driven three-dimensional chromatin structure for molecular classification in pancreatic cancers.

The molecular subtypes of pancreatic cancer (PC), either classical/progenitor-like or basal/squamous-like, are currently a major topic of research because of their direct association with clinical outcomes. Some transcription factors (TFs) have been reported to be associated with these subtypes. However, the mechanisms by which these molecular signatures of PCs are established remain unknown. Epigenetic regulatory processes, supported by dynamic changes in the chromatin structure, are essential for transcriptional profiles. Previously, we reported the importance of open chromatin profiles in the biological features and transcriptional status of PCs. Here, we aimed to analyze the relationships between three-dimensional (3D) genome structures and the molecular subtypes of human PCs using Hi-C analysis. We observed a correlation of the specific elements of 3D genome modules, including compartments, topologically associating domains, and enhancer-promoter loops, with the expression of related genes. We focused on HNF1B, a TF that is implicated in the progenitor subtype. Forced expression of HNF1B in squamous-type PC organoids induced the upregulation and downregulation of genes associated with progenitor and squamous subtypes, respectively. Long-range genomic interactions induced by HNF1B were accompanied by compartment modulation and H3K27ac redistribution. We also found that these HNF1B-induced changes in subtype-related gene expression required an intrinsically disordered region, suggesting a possible involvement of phase separation in compartment modulation. Thus, mapping of 3D structural changes induced by TFs, such as HNF1B, may become a useful resource for further understanding the molecular features of PCs.

Inhibiting SCAP/SREBP exacerbates liver injury and carcinogenesis in murine nonalcoholic steatohepatitis.

Enhanced de novo lipogenesis mediated by sterol regulatory element-binding proteins (SREBPs) is thought to be involved in nonalcoholic steatohepatitis (NASH) pathogenesis. In this study, we assessed the impact of SREBP inhibition on NASH and liver cancer development in murine models. Unexpectedly, SREBP inhibition via deletion of the SREBP cleavage-activating protein (SCAP) in the liver exacerbated liver injury, fibrosis, and carcinogenesis despite markedly reduced hepatic steatosis. These phenotypes were ameliorated by restoring SREBP function. Transcriptome and lipidome analyses revealed that SCAP/SREBP pathway inhibition altered the fatty acid (FA) composition of phosphatidylcholines due to both impaired FA synthesis and disorganized FA incorporation into phosphatidylcholine via lysophosphatidylcholine acyltransferase 3 (LPCAT3) downregulation, which led to endoplasmic reticulum (ER) stress and hepatocyte injury. Supplementation with phosphatidylcholines significantly improved liver injury and ER stress induced by SCAP deletion. The activity of the SCAP/SREBP/LPCAT3 axis was found to be inversely associated with liver fibrosis severity in human NASH. SREBP inhibition also cooperated with impaired autophagy to trigger liver injury. Thus, excessively strong and broad lipogenesis inhibition was counterproductive for NASH therapy; this will have important clinical implications in NASH treatment.

MNX1-HNF1B Axis Is Indispensable for Intraductal Papillary Mucinous Neoplasm Lineages.

BACKGROUND & AIMS: Chromatin architecture governs cell lineages by regulating the specific gene expression; however, its role in the diversity of cancer development remains unknown. Among pancreatic cancers, pancreatic ductal adenocarcinoma (PDAC) and intraductal papillary mucinous neoplasms (IPMN) with an associated invasive carcinoma (IPMNinv) arise from 2 distinct precursors, and their fundamental differences remain obscure. Here, we aimed to assess the difference of chromatin architecture regulating the transcriptional signatures or biological features in pancreatic cancers. METHODS: We established 28 human organoids from distinct subtypes of pancreatic tumors, including IPMN, IPMNinv, and PDAC. We performed exome sequencing (seq), RNA-seq, assay for transposase-accessible chromatin-seq, chromatin immunoprecipitation-seq, high-throughput chromosome conformation capture, and phenotypic analyses with short hairpin RNA or clustered regularly interspaced short palindromic repeats interference. RESULTS: Established organoids successfully reproduced the histology of primary tumors. IPMN and IPMNinv organoids harbored GNAS, RNF43, or KLF4 mutations and showed the distinct expression profiles compared with PDAC. Chromatin accessibility profiles revealed the gain of stomach-specific open regions in IPMN and the pattern of diverse gastrointestinal tissues in IPMNinv. In contrast, PDAC presented an impressive loss of accessible regions compared with normal pancreatic ducts. Transcription factor footprint analysis and functional assays identified that MNX1 and HNF1B were biologically indispensable for IPMN lineages. The upregulation of MNX1 was specifically marked in the human IPMN lineage tissues. The MNX1-HNF1B axis governed a set of genes, including MYC, SOX9, and OLFM4, which are known to be essential for gastrointestinal stem cells. High-throughput chromosome conformation capture analysis suggested the HNF1B target genes to be 3-dimensionally connected in the genome of IPMNinv. CONCLUSIONS: Our organoid analyses identified the MNX1-HNF1B axis to be biologically significant in IPMN lineages.

TET1 upregulation drives cancer cell growth through aberrant enhancer hydroxymethylation of HMGA2 in hepatocellular carcinoma.

Ten-eleven translocation 1 (TET1) is an essential methylcytosine dioxygenase of the DNA demethylation pathway. Despite its dysregulation being known to occur in human cancer, the role of TET1 remains poorly understood. In this study, we report that TET1 promotes cell growth in human liver cancer. The transcriptome analysis of 68 clinical liver samples revealed a subgroup of TET1-upregulated hepatocellular carcinoma (HCC), demonstrating hepatoblast-like gene expression signatures. We performed comprehensive cytosine methylation and hydroxymethylation (5-hmC) profiling and found that 5-hmC was aberrantly deposited preferentially in active enhancers. TET1 knockdown in hepatoma cell lines decreased hmC deposition with cell growth suppression. HMGA2 was highly expressed in a TET1high subgroup of HCC, associated with the hyperhydroxymethylation of its intronic region, marked as histone H3K4-monomethylated, where the H3K27-acetylated active enhancer chromatin state induced interactions with its promoter. Collectively, our findings point to a novel type of epigenetic dysregulation, methylcytosine dioxygenase TET1, which promotes cell proliferation via the ectopic enhancer of its oncogenic targets, HMGA2, in hepatoblast-like HCC.

Immunomodulation by Inflammation during Liver and Gastrointestinal Tumorigenesis and Aging.

Chronic inflammation is thought to promote tumorigenesis and metastasis by several mechanisms, such as affecting tumor cells directly, establishing a tumor-supporting microenvironment, enhancing tumor angiogenesis, and suppressing antitumor immunity. In this review, we discuss the recent advances in our understanding of how inflammation induces the immunosuppressive tumor microenvironment, such as increasing the level of pro-inflammatory cytokines, chemokines, and immunosuppressive molecules, inducing immune checkpoint molecules and cytotoxic T-cell exhaustion, and accumulating regulatory T (Treg) cells and myeloid-derived suppressor cells (MDSCs). The suppression of antitumor immunity by inflammation is especially examined in the liver and colorectal cancer. In addition, chronic inflammation is induced during aging and causes age-related diseases, including cancer, by affecting immunity. Therefore, we also discuss the age-related diseases regulated by inflammation, especially in the liver and colon.

Post-treatment cell-free DNA as a predictive biomarker in molecular-targeted therapy of hepatocellular carcinoma.

BACKGROUND: Liquid biopsies, particularly those involving circulating tumor DNA (ctDNA), are rapidly emerging as a non-invasive alternative to tumor biopsies. However, clinical applications of ctDNA analysis in hepatocellular carcinoma (HCC) have not been fully elucidated. METHODS: We measured the amount of plasma-derived cell-free DNA (cfDNA) in HCC patients before (n = 100) and a few days after treatment (n = 87), including radiofrequency ablation, transarterial chemoembolization, and molecular-targeted agents (MTAs), and prospectively analyzed their associations with clinical parameters and prognosis. TERT promoter mutations in cfDNA were analyzed using droplet digital PCR. Furthermore, we performed a comprehensive mutational analysis of post-treatment cfDNA via targeted ultra-deep sequencing (22,000× coverage) in a panel of 275 cancer-related genes in selected patients. RESULTS: Plasma cfDNA levels increased significantly according to HCC clinical stage, and a high cfDNA level was independently associated with a poor prognosis. TERT promoter mutations were detected in 45% of all cases but were not associated with any clinical characteristics. cfDNA levels increased significantly a few days after treatment, and a greater increase in post-treatment cfDNA levels was associated with a greater therapeutic response to MTAs. The detection rate of TERT mutations increased to 57% using post-treatment cfDNA, suggesting that the ctDNA was enriched. Targeted ultra-deep sequencing using post-treatment cfDNA after administering lenvatinib successfully detected various gene mutations and obtained promising results in lenvatinib-responsive cases. CONCLUSIONS: Post-treatment cfDNA analysis may facilitate the construction of biomarkers for predicting MTA treatment effects.

Inhibition of histone methyltransferase G9a attenuates liver cancer initiation by sensitizing DNA-damaged hepatocytes to p53-induced apoptosis.

While the significance of acquired genetic abnormalities in the initiation of hepatocellular carcinoma (HCC) has been established, the role of epigenetic modification remains unknown. Here we identified the pivotal role of histone methyltransferase G9a in the DNA damage-triggered initiation of HCC. Using liver-specific G9a-deficient (G9aΔHep) mice, we revealed that loss of G9a significantly attenuated liver tumor initiation caused by diethylnitrosamine (DEN). In addition, pharmacological inhibition of G9a attenuated the DEN-induced initiation of HCC. After treatment with DEN, while the induction of γH2AX and p53 were comparable in the G9aΔHep and wild-type livers, more apoptotic hepatocytes were detected in the G9aΔHep liver. Transcriptome analysis identified Bcl-G, a pro-apoptotic Bcl-2 family member, to be markedly upregulated in the G9aΔHep liver. In human cultured hepatoma cells, a G9a inhibitor, UNC0638, upregulated BCL-G expression and enhanced the apoptotic response after treatment with hydrogen peroxide or irradiation, suggesting an essential role of the G9a-Bcl-G axis in DNA damage response in hepatocytes. The proposed mechanism was that DNA damage stimuli recruited G9a to the p53-responsive element of the Bcl-G gene, resulting in the impaired enrichment of p53 to the region and the attenuation of Bcl-G expression. G9a deletion allowed the recruitment of p53 and upregulated Bcl-G expression. These results demonstrate that G9a allows DNA-damaged hepatocytes to escape p53-induced apoptosis by silencing Bcl-G, which may contribute to the tumor initiation. Therefore, G9a inhibition can be a novel preventive strategy for HCC.

Stromal SOX2 Upregulation Promotes Tumorigenesis through the Generation of a SFRP1/2-Expressing Cancer-Associated Fibroblast Population.

Cancer-associated fibroblasts (CAFs) promote tumor malignancy, but the precise transcriptional mechanisms regulating the acquisition of the CAF phenotype are not well understood. We show that the upregulation of SOX2 is central to this process, which is repressed by protein kinase Cζ (PKCζ). PKCζ deficiency activates the reprogramming of colonic fibroblasts to generate a predominant SOX2-dependent CAF population expressing the WNT regulator Sfrp2 as its top biomarker. SOX2 directly binds the Sfrp1/2 promoters, and the inactivation of Sox2 or Sfrp1/2 in CAFs impaired the induction of migration and invasion of colon cancer cells, as well as their tumorigenicity in vivo. Importantly, recurrence-free and overall survival of colorectal cancer (CRC) patients negatively correlates with stromal PKCζ levels. Also, SOX2 expression in the stroma is associated with CRC T invasion and worse prognosis of recurrence-free survival. Therefore, the PKCζ-SOX2 axis emerges as a critical step in the control of CAF pro-tumorigenic potential.

An Orthotopic Implantation Mouse Model of Hepatocellular Carcinoma with Underlying Liver Steatosis.

This protocol provides the steps required for a mouse liver orthotopic implantation model. The reliable pre-clinical animal models that have similar characteristics to hepatocellular carcinoma (HCC) are a powerful tool to unveil the mechanisms controlling tumor initiation and progression. Here, we describe a syngeneic orthotopic HCC model that recapitulates the role of a host pro-tumorigenic microenvironment by pre-conditioning mouse livers with a high-fat diet (HFD). For complete details on the use and execution of this protocol, please refer to Kudo et al. (2020).

Soluble VCAM-1 promotes gemcitabine resistance via macrophage infiltration and predicts therapeutic response in pancreatic cancer.

Pancreatic cancer is one of the malignant diseases with the worst prognosis. Resistance to chemotherapy is a major difficulty in treating the disease. We analyzed plasma samples from a genetically engineered mouse model of pancreatic cancer and found soluble vascular cell adhesion molecule-1 (sVCAM-1) increases in response to gemcitabine treatment. VCAM-1 was expressed and secreted by murine and human pancreatic cancer cells. Subcutaneous allograft tumors with overexpression or knock-down of VCAM-1, as well as VCAM-1-blocking treatment in the spontaneous mouse model of pancreatic cancer, revealed that sVCAM-1 promotes tumor growth and resistance to gemcitabine treatment in vivo but not in vitro. By analyzing allograft tumors and co-culture experiments, we found macrophages were attracted by sVCAM-1 to the tumor microenvironment and facilitated resistance to gemcitabine in tumor cells. In a clinical setting, we found that the change of sVCAM-1 in the plasma of patients with advanced pancreatic cancer was an independent prognostic factor for gemcitabine treatment. Collectively, gemcitabine treatment increases the release of sVCAM-1 from pancreatic cancer cells, which attracts macrophages into the tumor, thereby promoting the resistance to gemcitabine treatment. sVCAM-1 may be a potent clinical biomarker and a potential target for the therapy in pancreatic cancer.

Deletion of Histone Methyltransferase G9a Suppresses Mutant Kras-driven Pancreatic Carcinogenesis.

BACKGROUND/AIM: The entire mechanisms by which epigenetic modifiers contribute to the development of pancreatic cancer remain unknown. Although the histone methyltransferase G9a is a promising target in human cancers, its role in pancreatic carcinogenesis has been under-studied. The aim of the study was to examine the role of G9a in pancreatic carcinogenesis by a gene-targeting mouse model. MATERIALS AND METHODS: We established pancreas-specific G9aflox/flox mice and crossed them with Ptf1aCre/; KrasG12D/+ (KC) mice, which spontaneously develop pancreatic cancer. The phenotypes of the resulting KC mice with G9a deletion were examined. We analyzed transcriptomic data by microarray and genome-wide chromatin accessibility by transposase-accessible chromatin using sequencing. We established pancreatic organoids from KC mice. RESULTS: G9a deficiency impaired the progression of pancreatic intraepithelial neoplasia (PanIN) and prolonged the survival of KC mice. The number of phosphorylated Erk-positive cells and Dclk1-positive cells, which are reported to be essential for the progression of PanIN, were decreased by G9a deletion. UNC0638, an inhibitor of G9a, suppressed the growth of organoids and increased global chromatin accessibility, especially around the regions including the protein phosphatase 2A genes. CONCLUSION: Thus, our study suggested the functional interaction of G9a, Dclk1 and Mapk pathway in the Kras-driven pancreatic carcinogenesis. The inhibition of G9a may suppress the initiation of oncogenic Kras-driven pancreatic carcinogenesis.

PKCλ/ι Loss Induces Autophagy, Oxidative Phosphorylation, and NRF2 to Promote Liver Cancer Progression.

Oxidative stress plays a critical role in liver tissue damage and in hepatocellular carcinoma (HCC) initiation and progression. However, the mechanisms that regulate autophagy and metabolic reprogramming during reactive oxygen species (ROS) generation, and how ROS promote tumorigenesis, still need to be fully understood. We show that protein kinase C (PKC) λ/ι loss in hepatocytes promotes autophagy and oxidative phosphorylation. This results in ROS generation, which through NRF2 drives HCC through cell-autonomous and non-autonomous mechanisms. Although PKCλ/ι promotes tumorigenesis in oncogene-driven cancer models, emerging evidence demonstrate that it is a tumor suppressor in more complex carcinogenic processes. Consistently, PKCλ/ι levels negatively correlate with HCC histological tumor grade, establishing this kinase as a tumor suppressor in liver cancer.

Mutant IDH1 confers resistance to energy stress in normal biliary cells through PFKP-induced aerobic glycolysis and AMPK activation.

Metabolism is a critical regulator of cell fate determination. Recently, the significance of metabolic reprogramming in environmental adaptation during tumorigenesis has attracted much attention in cancer research. Recurrent mutations in the isocitrate dehydrogenase (IDH) 1 or 2 genes have been identified in several cancers, including intrahepatic cholangiocarcinoma (ICC). Mutant IDHs convert α-ketoglutarate (α-KG) to 2-hydroxyglutarate (2-HG), which affects the activity of multiple α-KG-dependent dioxygenases including histone lysine demethylases. Although mutant IDH can be detected even in the early stages of neoplasia, how IDH mutations function as oncogenic drivers remains unclear. In this study, we aimed to address the biological effects of IDH1 mutation using intrahepatic biliary organoids (IBOs). We demonstrated that mutant IDH1 increased the formation of IBOs as well as accelerated glucose metabolism. Gene expression analysis and ChIP results revealed the upregulation of platelet isoform of phosphofructokinase-1 (PFKP), which is a rate-limiting glycolytic enzyme, through the alteration of histone modification. Knockdown of the Pfkp gene alleviated the mutant IDH1-induced increase in IBO formation. Notably, the high expression of PFKP was observed more frequently in patients with IDH-mutant ICC compared to in those with wild-type IDH (p < 0.01, 80.9% vs. 42.5%, respectively). Furthermore, IBOs expressing mutant IDH1 survived the suppression of ATP production caused by growth factor depletion and matrix detachment by retaining high ATP levels through 5' adenosine monophosphate-activated protein kinase (AMPK) activation. Our findings provide a systematic understanding as to how mutant IDH induces tumorigenic preconditioning by metabolic rewiring in intrahepatic cholangiocytes.

CPT2 downregulation adapts HCC to lipid-rich environment and promotes carcinogenesis via acylcarnitine accumulation in obesity.

OBJECTIVE: Metabolic reprogramming of tumour cells that allows for adaptation to their local environment is a hallmark of cancer. Interestingly, obesity-driven and non-alcoholic steatohepatitis (NASH)-driven hepatocellular carcinoma (HCC) mouse models commonly exhibit strong steatosis in tumour cells as seen in human steatohepatitic HCC (SH-HCC), which may reflect a characteristic metabolic alteration. DESIGN: Non-tumour and HCC tissues obtained from diethylnitrosamine-injected mice fed either a normal or a high-fat diet (HFD) were subjected to comprehensive metabolome analysis, and the significance of obesity-mediated metabolic alteration in hepatocarcinogenesis was evaluated. RESULTS: The extensive accumulation of acylcarnitine species was seen in HCC tissues and in the serum of HFD-fed mice. A similar increase was found in the serum of patients with NASH-HCC. The accumulation of acylcarnitine could be attributed to the downregulation of carnitine palmitoyltransferase 2 (CPT2), which was also seen in human SH-HCC. CPT2 downregulation induced the suppression of fatty acid ß-oxidation, which would account for the steatotic changes in HCC. CPT2 knockdown in HCC cells resulted in their resistance to lipotoxicity by inhibiting the Src-mediated JNK activation. Additionally, oleoylcarnitine enhanced sphere formation by HCC cells via STAT3 activation, suggesting that acylcarnitine accumulation was a surrogate marker of CPT2 downregulation and directly contributed to hepatocarcinogenesis. HFD feeding and carnitine supplementation synergistically enhanced HCC development accompanied by acylcarnitine accumulation in vivo. CONCLUSION: In obesity-driven and NASH-driven HCC, metabolic reprogramming mediated by the downregulation of CPT2 enables HCC cells to escape lipotoxicity and promotes hepatocarcinogenesis.

Transparency-enhancing technology allows three-dimensional assessment of gastrointestinal mucosa: A porcine model.

Although high-resolution three-dimensional imaging of endoscopically resected gastrointestinal specimens can help elucidating morphological features of gastrointestinal mucosa or tumor, there are no established methods to achieve this without breaking specimens apart. We evaluated the utility of transparency-enhancing technology for three-dimensional assessment of gastrointestinal mucosa in porcine models. Esophagus, stomach, and colon mucosa samples obtained from a sacrificed swine were formalin-fixed and paraffin-embedded, and subsequently deparaffinized for analysis. The samples were fluorescently stained, optically cleared using transparency-enhancing technology: ilLUmination of Cleared organs to IDentify target molecules method (LUCID), and visualized using laser scanning microscopy. After observation, all specimens were paraffin-embedded again and evaluated by conventional histopathological assessment to measure the impact of transparency-enhancing procedures. As a result, microscopic observation revealed horizontal section views of mucosa at deeper levels and enabled the three-dimensional image reconstruction of glandular and vascular structures. Besides, paraffin-embedded specimens after transparency-enhancing procedures were all assessed appropriately by conventional histopathological staining. These results suggest that transparency-enhancing technology may be feasible for clinical application and enable the three-dimensional structural analysis of endoscopic resected specimen non-destructively. Although there remain many limitations or problems to be solved, this promising technology might represent a novel histopathological method for evaluating gastrointestinal cancers.

Stromal remodeling by the BET bromodomain inhibitor JQ1 suppresses the progression of human pancreatic cancer.

Inhibitors of bromodomain and extraterminal domain (BET) proteins, a family of chromatin reader proteins, have therapeutic efficacy against various malignancies. However, the detailed mechanisms underlying the anti-tumor effects in distinct tumor types remain elusive. Here, we show a novel antitumor mechanism of BET inhibition in pancreatic ductal adenocarcinoma (PDAC). We found that JQ1, a BET inhibitor, decreased desmoplastic stroma, a hallmark of PDAC, and suppressed the growth of patient-derived tumor xenografts (PDX) of PDACs. In vivo antitumor effects of JQ1 were not always associated with the JQ1 sensitivity of respective PDAC cells, and were rather dependent on the suppression of tumor-promoting activity in cancer-associated fibroblasts (CAFs). JQ1 inhibited Hedgehog and TGF-ß pathways as potent regulators of CAF activation and suppressed the expression of α-SMA, extracellular matrix, cytokines, and growth factors in human primary CAFs. Consistently, conditioned media (CM) from CAFs promoted the proliferation of PDAC cells along with the activation of ERK, AKT, and STAT3 pathways, though these effects were suppressed when CM from JQ1-treated CAFs was used. Mechanistically, chromatin immunoprecipitation experiments revealed that JQ1 reduced TGF-ß-dependent gene expression by disrupting the recruitment of the transcriptional machinery containing BET proteins. Finally, combination therapy with gemcitabine plus JQ1 showed greater efficacy than gemcitabine monotherapy against PDAC in vivo. Thus, our results reveal BET proteins as the critical regulators of CAF-activation and also provide evidence that stromal remodeling by epigenetic modulators can be a novel therapeutic option for PDAC.

A novel mouse model of intrahepatic cholangiocarcinoma induced by liver-specific Kras activation and Pten deletion.

Intrahepatic cholangiocarcinoma (ICC) is an aggressive malignancy with poor prognosis and its incidence is increasing worldwide. Recently, several types of cells have been considered as the origin of ICC, namely cholangiocytes, liver progenitor cells, and hepatocytes. Here, we have established a novel mouse model of ICC by liver-specific Kras activation and Pten deletion. An activating mutation of Kras in combination with deletion of Pten was introduced in embryonic hepatic bipotential progenitor cells (so-called hepatoblasts) and mature hepatocytes using the Cre-loxP system. As a result, liver-specific Kras activation and homozygous Pten deletion cooperated to induce ICCs exclusively. In contrast, Kras activation in combination with heterozygous Pten deletion induced both ICCs and HCCs, whereas Kras activation alone resulted in HCCs but not ICCs. Furthermore, a cell-lineage visualization system using tamoxifen-inducible Cre-loxP demonstrated that the ICCs did not originate from hepatocytes but from cholangiocytes. Our data suggest that mice carrying liver-specific Kras activation in combination with homozygous Pten deletion should be useful for the investigation of therapeutic strategies for human ICC.

A potent therapeutics for gallbladder cancer by combinatorial inhibition of the MAPK and mTOR signaling networks.

BACKGROUND: Gallbladder cancer (GBC) is the most common type of cancer with the worst prognosis among the bile duct cancers. There still remains a clear need for effective mechanism-based novel therapeutic approaches. A crosstalk between mitogen-activated protein kinase (MAPK) and the mammalian target of Rapamycin (mTOR) signaling pathways has been reported in several cancers. We hypothesized that targeting both pathways in combination will be a potent therapeutic for GBC. METHODS: Expression of phospho-ERK and phospho-S6rp protein were evaluated by immunostaining in surgically resected GBC specimens (n = 30). GBC cell lines and a xenograft model were treated with CI-1040, an inhibitor of MEK (mitogen-activated protein kinase kinase) and RAD001, an inhibitor of mTOR, alone or in combination, and then, we examined the cell proliferation and tumor growth, cell cycle status, and apoptosis. RESULTS: Analysis of human GBC tissues demonstrated that MAPK and mTOR signaling pathways were frequently coordinately dysregulated in one third of them. The combination therapy inhibited both signaling pathways and subsequently inhibited human GBC cell proliferation in vitro and xenograft tumor growth in vivo. Compared to the single treatment, the combination therapy significantly induced cell cycle arrest and apoptosis with decreased cyclin D1 expression. CONCLUSIONS: The double blockade of MAPK and mTOR signaling pathways inhibits the signal crosstalk and shows anti-tumor activity, which can be a potent therapeutic for GBC, especially for the patients with hyperactivated signaling of both pathways.