NNMT depletion contributes to liver cancer cell survival by enhancing autophagy under nutrient starvation.

Nicotinamide N-methyl transferase (NNMT) transfers a methyl group from S-adenosyl-L-methionine (SAM) to nicotinamide (NAM), producing 1-methylnicotinamide (1MNA). NNMT has been implicated in several cancer types and recently in metabolism, but its role in autophagy regulation has not yet been investigated. In this study, we determined that NNMT negatively regulated autophagy at the stage of ULK1 activation through protein phosphatase 2A (PP2A) activity. Specifically, NNMT knockdown increased PP2A methylation and subsequently enhanced phosphatase activity. Consequent p-ULK1 (S638) dephosphorylation derepressed ULK1 activity, resulting in autophagy induction. Accordingly, NNMT downregulation rescued tumor cells under nutrient deficiency in vivo, which was alleviated by ULK1 inhibitor treatment. In summary, our results suggest a novel mechanism by which tumor cells protect themselves against nutrient deprivation through NNMT suppression to accelerate autophagy.

Comparative Interactomes of VRK1 and VRK3 with Their Distinct Roles in the Cell Cycle of Liver Cancer.

Vaccinia-related kinase 1 (VRK1) and VRK3 are members of the VRK family of serine/threonine kinases and are principally localized in the nucleus. Despite the crucial roles of VRK1/VRK3 in physiology and disease, the molecular and functional interactions of VRK1/VRK3 are poorly understood. Here, we identified over 200 unreported VRK1/VRK3-interacting candidate proteins by affinity purification and LC-MS/MS. The networks of VRK1 and VRK3 interactomes were found to be associated with important biological processes such as the cell cycle, DNA repair, chromatin assembly, and RNA processing. Interactions of interacting proteins with VRK1/VRK3 were confirmed by biochemical assays. We also found that phosphorylations of XRCC5 were regulated by both VRK1/VRK3, and that of CCNB1 was regulated by VRK3. In liver cancer cells and tissues, VRK1/VRK3 were highly upregulated and its depletion affected cell cycle progression in the different phases. VRK3 seemed to affect S phase progression and G2 or M phase entry and exit, whereas VRK1 affects G1/S transition in the liver cancer, which could be explained by different interacting candidate proteins. Thus, this study not only provides a resource for investigating the unidentified functions of VRK1/VRK3, but also an insight into the regulatory roles of VRK1/VRK3 in biological processes.

Identification of Caveolin-1 as an Invasion-Associated Gene in Liver Cancer Cells Using Dendron-Coated DNA Microarrays.

The conventional gene expression profiling approaches have been replaced with DNA microarrays with exhibiting a powerful high-throughput capacity. Most solid surfaces of DNA microarrays contain such a high area density of functional groups to immobilize capture DNAs to the surface that the hybridization of capture DNAs with cDNA can be hindered, resulting in low intensity and reproducibility. Since our previous works showed that the 9-acid dendron was able to increase the hybridization efficiency, we aimed to demonstrate the feasibility of 9-acid dendron-coated glass slides as an advanced microarray platform for gene expression profiling. The 9-acid dendron-coated DNA microarray could reproducibly obtain the expression levels of 2800 human cancer-associated genes in the two liver cancer lines: Hep3B and SK-Hep1. Among the differentially expressed genes, Caveolin-1 (Cav-1) was identified as the most highly up-regulated gene in invasive SK-Hep1 in comparison to non-motile Hep3B. The overexpression of Cav-1 in Hep3B promoted the cell invasion, whereas its knockdown in SK-Hep1 suppressed the invasive feature, which confirms that the overexpression of Cav-1 is closely associated with cell invasion of liver carcinoma. Collectively, the 9-acid dendron-coated surface could successfully detect the transcript levels of cells, demonstrating its feasible potential to identify the candidate genes for further functional studies or diagnosis of diseases.

SIRT6 Depletion Suppresses Tumor Growth by Promoting Cellular Senescence Induced by DNA Damage in HCC.

The role of Sirtuin 6 (SIRT6) as a tumor suppressor or oncogene in liver cancer remains controversial. Thus, we identified the specific role of SIRT6 in the progression of hepatocellular carcinoma (HCC). SIRT6 expression was significantly higher in HCC cell lines and HCC tissues from 138 patients than in an immortalized hepatocyte cell line, THLE-2 and non-tumor tissues, respectively. SIRT6 knockdown by shRNA suppressed the growth of HCC cells and inhibited HCC tumor growth in vivo. In addition, SIRT6 silencing significantly prevented the growth of HCC cell lines by inducing cellular senescence in the p16/Rb- and p53/p21-pathway independent manners. Microarray analysis revealed that the expression of genes involved in nucleosome assembly was apparently altered in SIRT6-depleted Hep3B cells. SIRT6 knockdown promoted G2/M phase arrest and downregulation of genes encoding histone variants associated with nucleosome assembly, which could be attributed to DNA damage. Taken together, our findings suggest that SIRT6 acts as a tumor promoter by preventing DNA damage and cellular senescence, indicating that SIRT6 represents a potential therapeutic target for the treatment of HCC.

Vaccinia-related kinase 1 promotes hepatocellular carcinoma by controlling the levels of cell cycle regulators associated with G1/S transition.

We identified the specific role of vaccinia-related kinase 1 (VRK1) in the progression of hepatocellular carcinoma (HCC) and evaluated its therapeutic and prognostic potential. VRK1 levels were significantly higher in HCC cell lines than a normal hepatic cell line, and were higher in HCC than non-tumor tissue. VRK1 knockdown inhibited the proliferation of SK-Hep1, SH-J1 and Hep3B cells; moreover, depletion of VRK1 suppressed HCC tumor growth in vivo. We also showed that VRK1 knockdown increased the number of G1 arrested cells by decreasing cyclin D1 and p-Rb while upregulating p21 and p27, and that VRK1 depletion downregulated phosphorylation of CREB, a transcription factor regulating CCND1. Additionally, we found that luteolin, a VRK1 inhibitor, suppressed HCC growth in vitro and in vivo, and that the aberrant VRK1 expression correlated with poor prognostic features of HCC. High levels of VRK1 were associated with shorter overall and disease-free survival and higher recurrence rates. Taken together, our findings suggest VRK1 may act as a tumor promoter by controlling the level of cell cycle regulators associated with G1/S transition and could potentially serve as a therapeutic target and/or prognostic biomarker for HCC.

AROS Is a Significant Biomarker for Tumor Aggressiveness in Non-cirrhotic Hepatocellular Carcinoma.

Despite a low risk of liver failure and preserved liver function, non-cirrhotic hepatocellular carcinoma (HCC) has a poor prognosis. In the current study, we evaluated an active regulator of SIRT1 (AROS) as a prognostic biomarker in non-cirrhotic HCC. mRNA levels of AROS were measured in tumor and non-tumor tissues obtained from 283 non-cirrhotic HCC patients. AROS expression was exclusively up-regulated in recurrent tissues from the non-cirrhotic HCC patients (P = 0.015) and also in tumor tissues irrespective of tumor stage (P < 0.001) or BCLC stage (P < 0.001). High mRNA levels of AROS were statistically significantly associated with tumor stage (P < 0.001), BCLC stage (P = 0.007), alpha fetoprotein (AFP) level (P = 0.013), microvascular invasion (P = 0.001), tumor size (P = 0.036), and portal vein invasion (P = 0.005). Kaplan-Meir curve analysis demonstrated that HCC patients with higher AROS levels had shorter disease-free survival (DFS) in both the short-term (P < 0.001) and long-term (P = 0.005) compared to those with low AROS. Cox regression analysis demonstrated that AROS is a significant predictor for DFS along with large tumor size, tumor multiplicity, vascular invasion, and poor tumor differentiation, which are the known prognostic factors. In conclusion, AROS is a significant biomarker for tumor aggressiveness in non-cirrhotic hepatocellular carcinoma.

Ursolic acid exerts anti-cancer activity by suppressing vaccinia-related kinase 1-mediated damage repair in lung cancer cells.

Many mitotic kinases have been targeted for the development of anti-cancer drugs, and inhibitors of these kinases have been expected to perform well for cancer therapy. Efforts focused on selecting good targets and finding specific drugs to target are especially needed, largely due to the increased frequency of anti-cancer drugs used in the treatment of lung cancer. Vaccinia-related kinase 1 (VRK1) is a master regulator in lung adenocarcinoma and is considered a key molecule in the adaptive pathway, which mainly controls cell survival. We found that ursolic acid (UA) inhibits the catalytic activity of VRK1 via direct binding to the catalytic domain of VRK1. UA weakens surveillance mechanisms by blocking 53BP1 foci formation induced by VRK1 in lung cancer cells, and possesses synergistic anti-cancer effects with DNA damaging drugs. Taken together, UA can be a good anti-cancer agent for targeted therapy or combination therapy with DNA damaging drugs for lung cancer patients.

Brazilin Isolated from Caesalpinia sappan suppresses nuclear envelope reassembly by inhibiting barrier-to-autointegration factor phosphorylation.

To date, many anticancer drugs have been developed by directly or indirectly targeting microtubules, which are involved in cell division. Although this approach has yielded many anticancer drugs, these drugs produce undesirable side effects. An alternative strategy is needed, and targeting mitotic exit may be one alternative approach. Localization of phosphorylated barrier-to-autointegration factor (BAF) to the chromosomal core region is essential for nuclear envelope compartment relocalization. In this study, we isolated brazilin from Caesalpinia sappan Leguminosae and demonstrated that it inhibited BAF phosphorylation in vitro and in vivo. Moreover, we demonstrated direct binding between brazilin and BAF. The inhibition of BAF phosphorylation induced abnormal nuclear envelope reassembly and cell death, indicating that perturbation of nuclear envelope reassembly could be a novel approach to anticancer therapy. We propose that brazilin isolated from C. sappan may be a new anticancer drug candidate that induces cell death by inhibiting vaccinia-related kinase 1-mediated BAF phosphorylation.

Luteolin suppresses cancer cell proliferation by targeting vaccinia-related kinase 1.

Uncontrolled proliferation, a major feature of cancer cells, is often triggered by the malfunction of cell cycle regulators such as protein kinases. Recently, cell cycle-related protein kinases have become attractive targets for anti-cancer therapy, because they play fundamental roles in cellular proliferation. However, the protein kinase-targeted drugs that have been developed so far do not show impressive clinical results and also display severe side effects; therefore, there is undoubtedly a need to investigate new drugs targeting other protein kinases that are critical in cell cycle progression. Vaccinia-related kinase 1 (VRK1) is a mitotic kinase that functions in cell cycle regulation by phosphorylating cell cycle-related substrates such as barrier-to-autointegration factor (BAF), histone H3, and the cAMP response element (CRE)-binding protein (CREB). In our study, we identified luteolin as the inhibitor of VRK1 by screening a small-molecule natural compound library. Here, we evaluated the efficacy of luteolin as a VRK1-targeted inhibitor for developing an effective anti-cancer strategy. We confirmed that luteolin significantly reduces VRK1-mediated phosphorylation of the cell cycle-related substrates BAF and histone H3, and directly interacts with the catalytic domain of VRK1. In addition, luteolin regulates cell cycle progression by modulating VRK1 activity, leading to the suppression of cancer cell proliferation and the induction of apoptosis. Therefore, our study suggests that luteolin-induced VRK1 inhibition may contribute to establish a novel cell cycle-targeted strategy for anti-cancer therapy.

Influence of preoperative transcatheter arterial chemoembolization on gene expression in the HIF-1α pathway in patients with hepatocellular carcinoma.

PURPOSE: Although transcatheter arterial chemoembolization (TACE) is the most common treatment option in patients with hepatocellular carcinoma (HCC), its clinical benefits remain still controversial. Since TACE induces hypoxic necrosis in tumors, hypoxia-inducible factor 1α (HIF-1α) could critically affect biology in residual tumors after TACE treatment and subsequent prognosis. However, HIF-1α and its prognostic relevance in TACE have rarely been examined in human specimens. In the current study, we investigated the prognosis and expression of genes regulated by HIF-1α in HCC patients receiving preoperative TACE for the first time. METHODS: In total, 35 patients with HCC (10 patients undergoing preoperative TACE) were retrospectively studied. The prognostic significance of TACE was analyzed using Kaplan-Meier and Cox regression models. Protein levels of HIF-1α and mRNA levels of HIF-1α-associated genes were examined using immunohistochemistry (IHC) and real-time RT-PCR, respectively. RESULTS: Preoperative TACE was significantly associated with increased 2-year recurrence rate (80 vs. 36 %, P = 0.00402) and shorter disease-free survival (DFS) time (11.9 vs. 35.7 months, P = 0.0182). TACE was an independent prognostic factor for recurrence (P = 0.007) and poor DFS (P = 0.010) in a multivariate analysis. Immunohistochemical staining revealed in vivo activation of HIF-1α in human specimens treated with TACE. Notably, protein levels of HIF-1α were significantly increased in TACE tissues demonstrated by IHC. Transcriptional targets of HIF-1α showed mRNA expression patterns consistent with activation of HIF-1α in TACE tissues. CONCLUSIONS: Our findings collectively demonstrate that preoperative TACE confers poor prognosis in HCC patients through activation of HIF-1α.

Actionable gene expression-based patient stratification for molecular targeted therapy in hepatocellular carcinoma.

BACKGROUND: The effectiveness of molecular targeted agents is modest in hepatocellular carcinoma (HCC). Efficacy of molecular targeted therapies has been better in cancer patients with high expression of actionable molecules defined as cognate target molecules. However, patient stratification based on the actionable molecules dictating the effectiveness of targeted drugs has remained understudied in HCC. EXPERIMENTAL DESIGN & RESULTS: Paired tumor and non-tumoral tissues derived from a total of 130 HCC patients were studied. Real-time RT-PCR was used to analyze the mRNA expression of actionable molecules in the tissues. mRNA levels of EGFR, VEGFR2, PDGFRß, FGFR1, and mTOR were up-regulated in tumors compared to non-tumors in 35.4, 42.3, 61.5, 24.6, and 50.0% of patients, respectively. Up-regulation of EGFR was observed at early stage and tended to gradually decrease toward late stages (BCLC stage A: 41.9%; B: 30.8%; C: 17.6%). Frequency of VEGFR2 expression in tumors at stage C was lower than that in the other stages (BCLC stage A: 45.9%; B: 41.0%; C: 29.4%). PDGFRß and mTOR were observed to be up-regulated in more than 50% of tumors in all the stages whereas FGFR1 was up-regulated in only about 20% of HCC irrespective of stages. A cluster analysis of actionable gene expression revealed that HCC can be categorized into different subtypes that predict the effectiveness of molecular targeted agents and combination therapies in clinical trials. Analysis of in vitro sensitivity to sorafenib demonstrated that HCC cells with up-regulation of PDGFRß and c-Raf mRNA are more susceptible to sorafenib treatment in a dose and time-dependent manner than cells with low expression of the genes. CONCLUSIONS: mRNA expression analysis of actionable molecules could provide the rationale for new companion diagnostics-based therapeutic strategies in the treatment of HCC.

BNIP3 is degraded by ULK1-dependent autophagy via MTORC1 and AMPK.

BNIP3 (BCL2/adenovirus E1B 19 kDa interacting protein 3) is an atypical BH3-only protein that is induced by hypoxia-inducible factor 1 (HIF1) under hypoxia. BNIP3 is primarily regulated at the transcriptional level. However, little is known about the underlying mechanism of BNIP3 degradation. In this study, we found that BNIP3 was downregulated when hypoxia was accompanied by amino acid starvation. The BNIP3 downregulation did not occur at the transcription level and was independent of HIF1A. BNIP3 was primarily degraded by the proteasome, but BNIP3 was subjected to both proteasomal and autophagic degradation in response to starvation. The autophagic degradation of BNIP3 was dependent on ATG7 and MAP1LC3. We determined that autophagic degradation of BNIP3 was specifically regulated by ULK1 via the MTOR-AMPK pathway. Moreover, we confirmed that BNIP3 could play a protective role in tumor cells under hypoxia, and the treatment with Torin1, an MTOR inhibitor, decreased the BNIP3 level and enhanced the death of hypoxic tumor cells.

Rapamycin inhibits both motility through down-regulation of p-STAT3 (S727) by disrupting the mTORC2 assembly and peritoneal dissemination in sarcomatoid cholangiocarcinoma.

Cholangiocarcinoma (CC) is a malignant epithelium neoplasm that originates from the bile epithelium and for which there are few therapeutic strategies. The mTOR pathway involved in many cellular processes was reported to be up-regulated in various cancers. We investigated the activation of the AKT/mTOR pathway in CC cell lines with different degrees of dedifferentiation and found that rapamycin could suppress the motility and the peritoneal dissemination of sarcomatoid SCK cells. Inhibition of the mTOR pathway with rapamycin decreased significantly the number of tumor nodules and prolonged the survival rates of nude mice inoculated with sarcomatoid CC cells. Prolonged treatments with rapamycin were found to disrupt the mTORC2 assembly and to reduce the phosphorylation of STAT3 at Ser 727. Rapamycin decreased both mRNA and protein levels of MMP2 and Twist1, which are regulated by STAT3 and associated with cancer metastasis. The overexpression of STAT3 S727A lacking the phosphorylation site resulted in significantly less sensitivity to rapamycin than the overexpression of STAT3 WT. Taken together, our results suggest that rapamycin could suppress the motility of sarcomatoid CC by down-regulating p-STAT3 (S727) through the impairment of mTORC2 assembly.

REST-dependent expression of TRF2 renders non-neuronal cancer cells resistant to DNA damage during oxidative stress.

REST is a neuronal gene silencing factor ubiquitously expressed in non-neuronal tissues. REST is additionally believed to serve as a tumor suppressor in non-neuronal cancers. Conversely, recent findings on REST-dependent tumorigenesis in non-neuronal cells consistently suggest a potential role of REST as a tumor promoter. Here, we have uncovered for the first time the mechanism by which REST contributes to cancer cell survival in non-neuronal cancers. We observed abundant expression of REST in various types of non-neuronal cancer cells compared to normal tissues. The delicate roles of REST were further evaluated in HCT116 and HeLa, non-neuronal cancer cell lines expressing REST. REST silencing resulted in decreased cell survival and activation of the DNA damage response (DDR) through a decrease in the level of TRF2, a telomere-binding protein. These responses were correlated with reduced colony formation ability and accelerated telomere shortening in cancer cells upon the stable knockdown of REST. Interestingly, REST was down-regulated under oxidative stress conditions via ubiquitin proteasome system, suggesting that sustainability of REST expression is critical to determine cell survival during oxidative stress in a tumor microenvironment. Our results collectively indicate that REST-dependent TRF2 expression renders cancer cells resistant to DNA damage during oxidative stress, and mechanisms to overcome oxidative stress, such as high levels of REST or the stress-resistant REST mutants found in specific human cancers, may account for REST-dependent tumorigenesis.

Obtusilactone B from Machilus Thunbergii targets barrier-to-autointegration factor to treat cancer.

Targeting specific molecules is a promising cancer treatment because certain types of cancer cells are dependent on specific oncogenes. This strategy led to the development of therapeutics that use monoclonal antibodies or small-molecule inhibitors. However, the continued development of novel molecular targeting inhibitors is required to target the various oncogenes associated with the diverse types and stages of cancer. Obtusilactone B is a butanolide derivative purified from Machilus thunbergii. In this study, we show that obtusilactone B functions as a small-molecule inhibitor that causes abnormal nuclear envelope dynamics and inhibits growth by suppressing vaccinia-related kinase 1 (VRK1)-mediated phosphorylation of barrier-to-autointegration factor (BAF). BAF is important in maintaining lamin integrity, which is closely associated with diseases that include cancer. Specific binding of obtusilactone B to BAF suppressed VRK1-mediated BAF phosphorylation and the subsequent dissociation of the nuclear envelope from DNA that allows cells to progress through the cell cycle. Obtusilactone B potently induced tumor cell death in vitro, indicating that specific targeting of BAF to block cell cycle progression can be an effective anticancer strategy. Our results demonstrate that targeting a major constituent of the nuclear envelope may be a novel and promising alternative approach to cancer treatment.

Synergistic effect of orientation and lateral spacing of protein G on an on-chip immunoassay.

The proper orientation and lateral spacing of antibody molecules are a crucial element for an on-chip immunoassay in which the antibody or its antigen-binding fragments are immobilized on a solid surface. We covalently immobilized a modified protein G (Cys-protein G: protein G with only an N-terminal cysteine) on a dendron-coated surface to control its orientation and lateral spacing simultaneously. The cysteine-specific immobilization of Cys-protein G through the N-terminal cysteine resulted in 2.2-fold higher binding efficiency of Cys-protein G to IgG(2a) capture antibody than its random immobilization via lysine residues. The lateral spacing of 3.2 nm due to the surface modification with the 9-acid dendron molecule contributed to a 1.5-fold increase in the antibody-binding ability of Cys-protein G. Topographic images of atomic force microscopy exhibited a uniform coverage of Cys-protein G molecules immobilized on the thiol-reactive 9-acid dendron surface and homogeneous distribution of antibody bound to Cys-protein G. In the sandwich immunoassay, the control of the orientation of Cys-protein G led to 10-fold higher detection capability for rIL-2 compared with the randomly oriented protein G. The synergistic advantage of the unidirectional orientation and homogeneous lateral spacing of Cys-protein Gs on the dendron-coated surface can be applied to the development of more sensitive and reproducible antibody microarrays.

Signal integration by Akt regulates CD8 T cell effector and memory differentiation.

During a T cell response, the effector CTL pool contains two cellular subsets: short-lived effector cells (SLECs), a majority of which are destined for apoptosis, and the memory precursor effector cells, which differentiate into memory cells. Understanding the mechanisms that govern the differentiation of memory CD8 T cells is of fundamental importance in the development of effective CD8 T cell-based vaccines. The strength and nature of TCR signaling, along with signals delivered by cytokines like IL-2 and IL-12, influence differentiation of SLECs and memory precursor effector cells. A central question is, how are signals emanating from multiple receptors integrated and interpreted to define the fate of effector CTLs? Using genetic and pharmacological tools, we have identified Akt as a signal integrator that links distinct facets of CTL differentiation to the specific signaling pathways of FOXO, mTOR, and Wnt/ß-catenin. Sustained Akt activation triggered by convergent extracellular signals evokes a transcription program that enhances effector functions, drives differentiation of terminal effectors, and diminishes the CTLs' potential to survive and differentiate into memory cells. Whereas sustained Akt activation severely impaired CD8 T cell memory and protective immunity, in vivo inhibition of Akt rescued SLECs from deletion and increased the number of memory CD8 T cells. Thus, the cumulative strength of convergent signals from signaling molecules such as TCR, costimulatory molecules, and cytokine receptors governs the magnitude of Akt activation, which in turn controls the generation of long-lived memory cells. These findings suggest that therapeutic modulation of Akt might be a strategy to augment vaccine-induced immunity.

Overexpression of renal tumor antigen is associated with tumor invasion and poor prognosis of hepatocellular carcinoma.

PURPOSE: The aim of this study was to investigate the roles of renal tumor antigen (RAGE) in the progression and clinical outcome of hepatocellular carcinoma (HCC). METHODS: RAGE mRNA levels in 350 cases of HCC were investigated by quantitative real-time reverse transcription polymerase chain reaction. We analyzed the relationship of RAGE mRNA level with clinicopathologic parameters and clinical outcome. To identify the possible role of RAGE on cellular invasion, we performed in vitro analyses using small interfering RNAs (siRNAs). RESULTS: RAGE mRNA level was significantly higher in HCC than in noncancerous hepatic tissues (P < 0.001). Overexpression of RAGE was significantly correlated with the presence of multiple tumors (P = 0.021), high alfa-fetoprotein level (P = 0.042), and advanced tumor stage (P = 0.016). Higher levels of RAGE expression were associated with significantly shorter overall survival time (P = 0.029). Knockdown of RAGE expression by siRNAs suppressed the invasive ability of HCC cells and the expression and secretion of matrix metalloproteinase-9 (MMP-9). We found that RAGE and MMP-9 expressions were correlated in HCCs, and furthermore, the combination of RAGE and MMP-9 expression was associated with the survival of patients (P = 0.0066). CONCLUSIONS: Our results suggest that RAGE may be important in tumor invasion and could be a potential predictor for the prognosis of HCC patients.

The expression of phospho-AKT1 and phospho-MTOR is associated with a favorable prognosis independent of PTEN expression in intrahepatic cholangiocarcinomas.

AKT1 signaling pathway is important for the regulation of protein synthesis and cell survival with implications in carcinogenesis. In this study, we explored the prognostic significance of AKT1 pathway in intrahepatic cholangiocarcinomas. We investigated the status of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), phosphorylated (p) AKT1 (p-AKT1), p-mammalian target of rapamycin (p-MTOR), p-p70 ribosomal protein S6 kinase (p-RPS6KB2) and p-eukaryotic initiation factor 4E-binding protein-1 (p-EIF4EBP1) in 101 intrahepatic cholangiocarcinomas by immunohistochemistry. Western blot analysis was performed to verify the expression levels of p-AKT1 and p-MTOR. The relationship of protein expression with clinicopathological data and the correlations of protein expression levels were explored. The overexpression of p-AKT1, p-MTOR, and PTEN was associated with a better survival in patients with intrahepatic cholangiocarcinoma (P=0.0137, 0.0194, and 0.0337, respectively). In a multivariate analysis, PTEN was an independent prognostic factor, and p-AKT1 showed tendency (P=0.032 and 0.051, respectively). The overexpression of p-MTOR was correlated with well-to-moderately differentiated tumors (P<0.001) and tumors without metastasis (P=0.046). Expression levels of the AKT1 signaling pathway proteins in this study showed positive correlations with each other, except for PTEN. Aberrant expressions of p-AKT1 and p-MTOR in intrahepatic cholangiocarcinoma were associated with a favorable prognosis, possibly in a PTEN-independent manner. Our results indicate that dysregulation of the AKT1 pathway may have an important role in the development of intrahepatic cholangiocarcinoma, but not necessarily in the progression of the disease.