Following the classification of hepatocellular nodules by the International Working Party in 1995 and further elaboration by the International Consensus Group for Hepatocellular Neoplasia in 2009, entities under the spectrum of hepatocellular nodules have been better characterized. Research work hence has been done to answer questions such as distinguishing high-grade dysplastic nodules from early hepatocellular carcinoma (HCC), delineating the tumor cell origin of HCC, identifying its prognostic markers, and subtyping hepatocellular adenomas. As a result, a copious amount of data at immunohistochemical and molecular levels has emerged. A panel of immunohistochemical markers including glypican-3, heat shock protein 70 and glutamine synthetase has been found to be of use in the diagnosis of small, well differentiated hepatocellular tumors and particularly of HCC. The use of liver fatty acid binding protein (L-FABP), β-catenin, glutamine synthetase, serum amyloid protein and C-reactive protein is found to be helpful in the subtyping of hepatocellular adenomas. The role of tissue biomarkers for prognostication in HCC and the use of biomarkers in subclassifying HCC based on tumor cell origin are also discussed.
Hepatocellular tumors; Immunohistochemical; Classification
Enhancer of zeste homolog 2 (EZH2), the histone methyltransferase of the Polycomb Repressive complex 2 catalyzing histone H3 lysine 27 tri-methylation (H3K27me3), is frequently up-regulated in human cancers. In this study, we identified the tumor suppressor Deleted in liver cancer 1 (DLC1) as a target of repression by EZH2-mediated H3K27me3. DLC1 is a GTPase-activating protein for Rho family proteins. Inactivation of DLC1 results in hyper-activated Rho/ROCK signaling and is implicated in actin cytoskeleton reorganization to promote cancer metastasis. By chromatin immunoprecipitation assay, we demonstrated that H3K27me3 was significantly enriched at the DLC1 promoter region of a DLC1-nonexpressing HCC cell line, MHCC97L. Depletion of EZH2 in MHCC97L by shRNA reduced H3K27me3 level at DLC1 promoter and induced DLC1 gene re-expression. Conversely, transient overexpression of GFP-EZH2 in DLC1-expressing Huh7 cells reduced DLC1 mRNA level with a concomitant enrichment of EZH2 on DLC1 promoter. An inverse relation between EZH2 and DLC1 expression was observed in the liver, lung, breast, prostate, and ovarian cancer tissues. Treating cancer cells with the EZH2 small molecular inhibitor, 3-Deazaneplanocin A (DZNep), restored DLC1 expression in different cancer cell lines, indicating that EZH2-mediated H3K27me3 epigenetic regulation of DLC1 was a common mechanism in human cancers. Importantly, we found that DZNep treatment inhibited HCC cell migration through disrupting actin cytoskeleton network, suggesting the therapeutic potential of DZNep in targeting cancer metastasis. Taken together, our study has shed mechanistic insight into EZH2-H3K27me3 epigenetic repression of DLC1 and advocated the significant pro-metastatic role of EZH2 via repressing tumor and metastasis suppressors.
Hepatic progenitor cells (HPCs) are bipotential cells residing in normal liver. Their proliferation is observed in reactive conditions of the liver and in primary liver cancers. The observation that some hepatocellular carcinomas (HCCs) express a biliary-like immunophenotype has led to the identification of HPCs in HCC. Accumulating evidence suggests that HPCs play a role as the cell of origin in a variety of primary liver cancers. This has led to the development of revolutionary concepts in hepatocarcinogenesis. In this article, the role and significance of HPCs in HCC, including its classification, are summarized and discussed.
Classification; Hepatic progenitor cells; Hepatocarcinogenesis; Hepatocellular carcinoma; Primary liver cancer
AMP-activated protein kinase (AMPK), a biological sensor for cellular energy status, has been shown to act upstream and downstream of known tumour suppressors. However, whether AMPK itself plays a tumor suppressor role in cancer remains unclear. Here, we found that the α2 catalytic subunit isoform of AMPK is significantly down-regulated in hepatocellular carcinoma (HCC). Clinicopathological analysis revealed that under-expression of AMPK-α2 was statistically associated with an undifferentiated cellular phenotype and poor patient prognosis. Loss of AMPK-α2 in HCC cells rendered them more tumorigenic than control cells both in vitro and in vivo. Mechanistically, ectopic expression of AMPK enhanced the acetylation and stability of p53 in HCC cells. The p53 deacetylase, SIRT1, was phosphorylated and inactivated by AMPK at Thr-344, promoting p53 acetylation and apoptosis of HCC cells. Taken together, our findings suggest that under-expression of AMPK is frequently observed in HCC, and that inactivation of AMPK promotes hepatocarcinogenesis by destabilising p53 in a SIRT1-dependent manner.
AMPK; SIRT1; HCC; p53; phosphorylation
Hepatocellular carcinoma (HCC) is a highly aggressive cancer that is linked to chronically dysregulated liver inflammation. However, appropriate immune responses can control HCC progression. Here we investigated the role and underlying mechanism of toll-like receptor 3 (TLR3) in HCC.
HCC cell death, and natural killer (NK) cell activation and cytotoxicity were assessed in vitro after treatment with the TLR3 ligand poly(I:C). The effect of TLR3 on the tumor parenchyma and infiltrating immune cells was investigated in a spontaneous liver tumor mouse model and a transplanted tumor mouse model (n = 3–9 mice per group). Immunohistochemistry and quantitative polymerase chain reaction were used to analyze tumor samples from 172 HCC patients. Paired t-tests and analysis of variance tests were used to calculate P-values. The relationship between TLR3 expression and survival was determined by the Kaplan–Meier univariate survival analysis and a log-rank test. All statistical tests were two-sided.
TLR3 activation increased cell death in the TLR3+ SNU182 HCC cell line (30.5% vs 8.5%, P = .03) and promoted NK-cell activation (32.6% vs 19.4%, P < .001) and cytotoxicity (relative fourfold increase, P = .03) in vitro. In vivo, poly(I:C) treatment increased intratumoral chemokine expression, NK-cell activation and tumor infiltration, and proliferation of tumor-infiltrating T and NK cells. Proliferation of tumor parenchyma cells was decreased. Also, expression of chemokines or treatment with poly(I:C) decreased tumor growth. TLR3 expression in patient samples correlated with NK-cell activation, NK- and T-cell tumor infiltration, and inversely correlated with tumor parenchyma cell viability. TLR3 expression was also associated with longer survival in HCC patients (hazard ratio of survival = 2.1, 95% confidence interval = 1.3 to 3.4, P = .002).
TLR3 is an important modulator of HCC progression and is a potential target for novel immunotherapy.
CDK5 regulatory subunit associated protein 3 (CDK5RAP3) is a novel activator of PAK4 and processes important pro-metastatic function in hepatocarcinogenesis. However, it remains unclear if there are other mechanisms by which CDK5RAP3 promotes HCC metastasis. Here, we showed that in CDK5RAP3 stable knockdown SMMC-7721 HCC cells, p14ARF tumor suppressor was upregulated at protein and mRNA levels, and ectopic expression of CDK5RAP3 was found to repress the transcription of p14ARF. Using chromatin immunoprecipitation assay, we demonstrated that CDK5RAP3 bound to p14ARF promoter in vivo. Furthermore, knockdown of p14ARF in CDK5RAP3 stable knockdown HCC cells reversed the suppression of HCC cell invasiveness mediated by knockdown of CDK5RAP3. Taken together, our findings provide the new evidence that overexpression of CDK5RAP3 promotes HCC metastasis via downregulation of p14ARF.
Recent evidence suggested that prostate cancer stem/progenitor cells (CSC) are responsible for cancer initiation as well as disease progression. Unfortunately, conventional therapies are only effective in targeting the more differentiated cancer cells and spare the CSCs. Here, we report that PSP, an active component extracted from the mushroom Turkey tail (also known as Coriolus versicolor), is effective in targeting prostate CSCs. We found that treatment of the prostate cancer cell line PC-3 with PSP led to the down-regulation of CSC markers (CD133 and CD44) in a time and dose-dependent manner. Meanwhile, PSP treatment not only suppressed the ability of PC-3 cells to form prostaspheres under non-adherent culture conditions, but also inhibited their tumorigenicity in vivo, further proving that PSP can suppress prostate CSC properties. To investigate if the anti-CSC effect of PSP may lead to prostate cancer chemoprevention, transgenic mice (TgMAP) that spontaneously develop prostate tumors were orally fed with PSP for 20 weeks. Whereas 100% of the mice that fed with water only developed prostate tumors at the end of experiment, no tumors could be found in any of the mice fed with PSP, suggesting that PSP treatment can completely inhibit prostate tumor formation. Our results not only demonstrated the intriguing anti-CSC effect of PSP, but also revealed, for the first time, the surprising chemopreventive property of oral PSP consumption against prostate cancer.
DNA methylation and histone modifications are two major epigenetic events regulating gene expression and chromatin structure, and their alterations are linked to human carcinogenesis. DNA methylation plays an important role in tumor suppressor gene inactivation, and can be revised by DNA methylation inhibitors. The reversible nature of DNA methylation forms the basis of epigenetic cancer therapy. However, it has been reported that DNA re-methylation and gene re-silencing could occur after removal of demethylation treatment and this may significantly hamper the therapeutic value of DNA methylation inhibitors. In this study we have provided detailed evidence demonstrating that mammalian cells possess a bona fide DNA methylation recovery system. We have also shown that DNA methylation recovery was mediated by the major human DNA methyltransferase, DNMT1. In addition, we found that H3K9-tri-methylation and H3K27-tri-methylation were closely associated with this DNA methylation recovery. These persistent transcriptional repressive histone modifications may have a crucial role in regulating DNMT1-mediated DNA methylation recovery. Our findings may have important implications towards a better understanding of epigenetic regulation and future development of epigenetic therapeutic intervention.
Diclofenac is one of the oldest anti-inflammatory drugs in use. In addition to its inhibition of cyclooxygenases (COX), diclofenac potently inhibits phospholipase A2 (PLA2), thus yielding a broad anti-inflammatory effect. Since inflammation is an important factor in the development of pancreatic tumors we explored the potential of diclofenac to inhibit tumor growth in mice inoculated with PANCO2 cells orthotopically.
We found that diclofenac treatment (30 mg/kg/bw for 11 days) of mice inoculated with PANC02 cells, reduced the tumor weight by 60%, correlating with increased apoptosis of tumor cells. Since this effect was not observed in vitro on cultured PANCO2 cells, we theorized that diclofenac beneficial treatment involved other mediators present in vivo. Indeed, diclofenac drastically decreased tumor vascularization by downregulating VEGF in the tumor and in abdominal cavity fluid. Furthermore, diclofenac directly inhibited vascular sprouting ex vivo. Surprisingly, in contrast to other COX-2 inhibitors, diclofenac increased arginase activity/arginase 1 protein content in tumor stroma cells, peritoneal macrophages and white blood cells by 2.4, 4.8 and 2 fold, respectively. We propose that the subsequent arginine depletion and decrease in NO levels, both in serum and peritoneal cavity, adds to tumor growth inhibition by malnourishment and poor vasculature development.
In conclusion, diclofenac shows pronounced antitumoral properties in pancreatic cancer model that can contribute to further treatment development. The ability of diclofenac to induce arginase activity in tumor stroma, peritoneal macrophages and white blood cells provides a tool to study a controversial issue of pro-and antitumoral effects of arginine depletion.
Nonalcoholic fatty liver disease (NAFLD) is a common form of chronic liver disease, and serum uric acid is observed to be significantly elevated in NAFLD patients. However, whether this elevation is causal, a bystander, or a consequence of NAFLD remains unclear. We performed a population-based prospective study among the employees of Zhenhai Refining & Chemical Company Ltd., Ningbo, China to investigate whether the elevation of serum uric acid has a casual role for NAFLD. A total of 6890 initially NAFLD-free subjects were followed up for 3 years. Overall, 11.80% (813/6890) subjects developed NAFLD over 3 years of follow-up. The cumulative incidence of NAFLD increased with progressively higher baseline serum uric acid levels (the cumulative incidence was 7.2%, 9.5%, 11.5%, 13.8%, and 17.2% in quintile 1, quintile 2, 3, 4 and 5, respectively; P value for trend <0.001). Cox proportional hazards regression analyses showed that serum uric acid levels were independently and positively associated with the risk for incident NAFLD; the age-, gender- and metabolic syndrome adjusted hazard ratio (95% CI) for the subjects in quintile 2, 3, 4 and 5 versus quintile 1 was 1.18 (0.91–1.54), 1.32 (1.03–1.70), 1.39 (1.09–1.78) and 1.50 (1.18–1.92), respectively. Taken together, our prospective observational study showed that elevation of serum uric acid levels independently predicts increase risk for incident NAFLD.
DLC2 (deleted in liver cancer 2), a Rho GTPase-activating protein, was previously shown to be underexpressed in human hepatocellular carcinoma and has tumor suppressor functions in cell culture models. We generated DLC2-deficient mice to investigate the tumor suppressor role of DLC2 in hepatocarcinogenesis and the function of DLC2 in vivo. In this study, we found that, unlike homologous DLC1, which is essential for embryonic development, DLC2 was dispensable for embryonic development and DLC2-deficient mice could survive to adulthood. We also did not observe a higher incidence of liver tumor formation or diethylnitrosamine (DEN)-induced hepatocarcinogenesis in DLC2-deficient mice. However, we observed that DLC2-deficient mice were smaller and had less adipose tissue than the wild type mice. These phenotypes were not due to reduction of cell size or defect in adipogenesis, as observed in the 190B RhoGAP-deficient mouse model. Together, these results suggest that deficiency in DLC2 alone does not enhance hepatocarcinogenesis.
Deleted in liver cancer 1 (DLC1) is a Rho GTPase-activating protein (RhoGAP) frequently deleted and underexpressed in hepatocellular carcinoma (HCC) as well as in other cancers. Recent independent studies have shown interaction of DLC1 with members of the tensin focal adhesion protein family in a Src Homology 2 (SH2) domain-dependent mechanism. DLC1 and tensins interact and co-localize to punctate structures at focal adhesions. However, the mechanisms underlying the interaction between DLC1 and various tensins remain controversial.
We used a co-immunoprecipitation assay to identify a previously undocumented binding site at 375–385 of DLC1 that predominantly interacted with the phosphotyrosine binding (PTB) domain of tensin2. DLC1-tensin2 interaction is completely abolished in a DLC1 mutant lacking this novel PTB binding site (DLC1ΔPTB). However, as demonstrated by immunofluorescence and co-immunoprecipitation, neither the focal adhesion localization nor the interaction with tensin1 and C-terminal tensin-like (cten) were affected. Interestingly, the functional significance of this novel site was exhibited by the partial reduction of the RhoGAP activity, which, in turn, attenuated the growth-suppressive activity of DLC1 upon its removal from DLC1.
This study has provided new evidence that DLC1 also interacts with tensin2 in a PTB domain-dependent manner. In addition to properly localizing focal adhesions and preserving RhoGAP activity, DLC1 interaction with tensin2 through this novel focal adhesion binding site contributes to the growth-suppressive activity of DLC1.
Deleted in liver cancer 1 (DLC1), a member of RhoGTPase activating protein (GAP) family, is known to have suppressive activities in tumorigenicity and cancer metastasis. However, the underlying molecular mechanisms of how DLC1 suppresses cell motility have not been fully elucidated. Rho-kinase (ROCK) is an immediate down-stream effector of RhoA in mediating cellular cytoskeletal events and cell motility. In the present study, we aimed to investigate the effects of DLC1 on Rho/ROCK signaling pathway in hepatocellular carcinoma (HCC).
We demonstrated that DLC1 negatively regulated ROCK-dependent actomyosin contractility. From immumofluorescence study, we found that ectopic expression of DLC1 abrogated Rho/ROCK-mediated cytoskeletal reorganization including formation of stress fibers and focal adhesions. It also downregulated cortical phosphorylation of myosin light chain 2 (MLC2). These inhibitory events by DLC1 were RhoGAP-dependent, as RhoGAP-deficient mutant of DLC1 (DLC1 K714E) abolished these inhibitory events. In addition, from western study, DLC1 inhibited ROCK-related myosin light chain phosphatase targeting unit 1 (MYPT1) phosphorylation at Threonine 853. By examining cell morphology under microscope, we found that ectopic expression of dominant-active ROCK released cells from DLC1-induced cytoskeletal collapse and cell shrinkage.
Our data suggest that DLC1 negatively regulates Rho/ROCK/MLC2. This implicates a ROCK-mediated pathway of DLC1 in suppressing metastasis of HCC cells and enriches our understanding in the molecular mechanisms involved in the progression of hepatocellular carcinoma.
We have previously characterized transcription factor LZIP to be a growth suppressor targeted by hepatitis C virus oncoprotein. In search of proteins closely related to LZIP, we have identified a liver-enriched transcription factor CREB-H. LZIP and CREB-H represent a new subfamily of bZIP factors. CREB-H activates transcription by binding to cAMP responsive element, box B, and ATF6-binding element. Interestingly, CREB-H has a putative transmembrane (TM) domain and it localizes ambiently to the endoplasmic reticulum. Proteolytic cleavage that removes the TM domain leads to nuclear translocation and activation of CREB-H. CREB-H activates the promoter of hepatic gluconeogenic enzyme phosphoenolpyruvate carboxykinase. This activation can be further stimulated by cAMP and protein kinase A. CREB-H transcript is exclusively abundant in adult liver. In contrast, the expression of CREB-H mRNA is aberrantly reduced in hepatoma tissues and cells. The enforced expression of CREB-H suppresses the proliferation of cultured hepatoma cells. Taken together, our findings suggest that the liver-enriched bZIP transcription factor CREB-H is a growth suppressor that plays a role in hepatic physiology and pathology.
Activation of the Wnt/β-catenin signaling pathway plays a crucial role in hepatocellular carcinoma (HCC). Low-density lipoprotein (LDL) receptor-related protein-6 (LRP6) is one of the co-receptors of the Wnt/β-catenin pathway and forms a signaling complex with Wnt ligand and Frizzled receptor to activate downstream signaling. However, the role of LRP6 in hepatocarcinogenesis is unclear. In this study, we examined its expression and roles in human HCC.
Using real-time quantitative RT-PCR, we found that LRP6 was frequently (45%) overexpressed in human HCCs (P = 0.003). In vitro studies showed that ectopic expression of LRP6 increased the protein level of β-catenin. Moreover, overexpression of the full-length and constitutively active LRP6, respectively, activated the WNT/β-catenin signaling pathway, as shown by the TCF/β-catenin reporter assay. With regard to the effects of LRP6 overexpression in HCC cells, stable overexpression of the constitutively active LRP6 in BEL-7402 HCC cells enhanced cell proliferation, cell migration, and invasion in vitro as well as tumorigenicity in nude mice.
Our findings indicate that overexpression of LRP6 contributes to the hyperactivation of the Wnt/β-catenin signaling pathway in human HCCs and suggest it may play a role in hepatocarcinogenesis.
One of the most relevant risk factors for hepatocellular carcinoma (HCC) development is chronic hepatitis B virus (HBV) infection, but only a fraction of chronic HBV carriers develop HCC, indicating that complex interactions among viral, environmental and genetic factors lead to HCC in HBV-infected patients. So far, host genetic factors have incompletely been characterized. Therefore, we performed a genome-wide association (GWA) study in a Southern Chinese cohort consisting of 95 HBV-infected HCC patients (cases) and 97 HBV-infected patients without HCC (controls) using the Illumina Human610-Quad BeadChips. The top single nucleotide polymorphisms (SNPs) were then validated in an independent cohort of 500 cases and 728 controls. 4 SNPs (rs12682266, rs7821974, rs2275959, rs1573266) at chromosome 8p12 showed consistent association in both the GWA and replication phases (ORcombined = 1.31–1.39; pcombined = 2.71×10−5–5.19×10−4; PARcombined = 26–31%). We found a 2.3-kb expressed sequence tag (EST) in the region using in-silico data mining and verified the existence of the full-length EST experimentally. The expression level of the EST was significantly reduced in human HCC tumors in comparison to the corresponding non-tumorous liver tissues (P<0.001). Results from sequence analysis and in-vitro protein translation study suggest that the transcript might function as a long non-coding RNA. In summary, our study suggests that variations at chromosome 8p12 may promote HCC in patients with HBV. Further functional studies of this region may help understand HBV-associated hepatocarcinogenesis.
Deleted in liver cancer 1 (DLC1) serves as an important RhoGTPase activating protein (RhoGAP) protein that terminates active RhoA signaling in human cancers. Increasing evidence has demonstrated that the tumor suppressive activity of DLC1 depends not only on RhoGAP activity, but also relies on proper focal adhesion localization through its interaction with tensin family proteins. Recently, there are reports showing that DLC1 can also be found in the nucleus; however, the existence and the relative tumor suppressive activity of nuclear DLC1 have never been clearly addressed.
Methodology and Principal Findings
We herein provide new evidence that DLC1 protein, which predominantly associated with focal adhesions and localized in cytosol, dynamically shuttled between cytoplasm and nucleus. Treatment of cells with nuclear export blocker, Leptomycin B (LMB), retained DLC1 in the nucleus. To understand the nuclear entry of DLC1, we identified amino acids 600–700 of DLC1 as a novel region that is important for its nuclear localization. The tumor suppressive activity of nuclear DLC1 was directly assessed by employing a nuclear localization signal (NLS) fusion variant of DLC1 (NLS-DLC1) with preferential nuclear localization. In SMMC-7721 HCC cells, expression of NLS-DLC1 failed to suppress colony formation and actin stress fiber formation in vitro. The abrogated tumor suppressive activity of nuclear DLC1 was demonstrated for the first time in vivo by subcutaneously injecting p53−/− RasV12 hepatoblasts with stable NLS-DLC1 expression in nude mice. The injected hepatoblasts with NLS-DLC1 expression effectively formed tumors when compared with the non-nuclear targeted DLC1.
Our study identified a novel region responsible for the nuclear entry of DLC1 and demonstrated the functional difference of DLC1 in different cellular compartments both in vitro and in vivo.
Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease characterized by late diagnosis and treatment resistance. Recurrent genetic alterations in defined genes in association with perturbations of developmental cell signaling pathways have been associated with PDAC development and progression. Here, we show that GATA6 contributes to pancreatic carcinogenesis during the temporal progression of pancreatic intraepithelial neoplasia by virtue of Wnt pathway activation. GATA6 is recurrently amplified by both quantitative-PCR and fluorescent in-situ hybridization in human pancreatic intraepithelial neoplasia and in PDAC tissues, and GATA6 copy number is significantly correlated with overall patient survival. Forced overexpression of GATA6 in cancer cell lines enhanced cell proliferation and colony formation in soft agar in vitro and growth in vivo, as well as increased Wnt signaling. By contrast siRNA mediated knockdown of GATA6 led to corresponding decreases in these same parameters. The effects of GATA6 were found to be due to its ability to bind DNA, as forced overexpression of a DNA-binding mutant of GATA6 had no effects on cell growth in vitro or in vivo, nor did they affect Wnt signaling levels in these same cells. A microarray analysis revealed the Wnt antagonist Dickopf-1 (DKK1) as a dysregulated gene in association with GATA6 knockdown, and direct binding of GATA6 to the DKK1 promoter was confirmed by chromatin immunoprecipitation and electrophoretic mobility shift assays. Transient transfection of GATA6, but not mutant GATA6, into cancer cell lines led to decreased DKK1 mRNA expression and secretion of DKK1 protein into culture media. Forced overexpression of DKK1 antagonized the effects of GATA6 on Wnt signaling in pancreatic cancer cells. These findings illustrate that one mechanism by which GATA6 promotes pancreatic carcinogenesis is by virtue of its activation of canonical Wnt signaling via regulation of DKK1.
To assess the clinical significance and prognostic impact of extranodal metastasis (EM) in gastric carcinoma and establish an optimal classification in the staging system.
A total of 1343 patients with gastric carcinoma who underwent surgical resection were recruited to determine the frequency and prognostic significance of EMs. EMs were divided into two groups (EM1 and EM2) and then incorporated into the 7th edition UICC TNM staging system. EMs was detected in 179 (13.3%) of 1343 patients who underwent radical resection. Multivariate analysis identified EMs as an independent prognostic factor (HR = 1.412, 95%CI = 1.151–1.731, P<0.001). After curative operation, the overall survival rate were worse in patients with ≥3 cases of EM (EM2) than those with the number of 1 and 2 cases (EM1) (P<0.001). Survival of patients with EM1 was found almost comparable to that of N3 stage (P = 0.437). Survival of patients with EM2 showed similar to that of stage IV patients (P = 0.896). By using the linear trend X2, likelihood ratio X2, and Akaike information criterion (AIC) test, EM1 treated as N3 stage and EM2 treated as M1 stage performed higher linear trend X2 scores, likelihood ratio X2 scores, and lower AIC value than the 7th edition UICC TNM staging system, which represented the optimum prognostic stratification, together with better homogeneity, discriminatory ability, and monotonicity of gradients.
EMs might be classified based on their number and prognostic information and should incorporate into the TNM staging system.
Surgical samples have long been used as important subjects for cancer research. In accordance with an increase of neoadjuvant therapy, biopsy samples have recently become imperative for cancer transcriptome. On the other hand, both biopsy and surgical samples are available for expression profiling for predicting clinical outcome by adjuvant therapy; however, it is still unclear whether surgical sample expression profiles are useful for prediction via biopsy samples, because little has been done about comparative gene expression profiling between the two kinds of samples.
Methodology and Findings
A total of 166 samples (77 biopsy and 89 surgical) of normal and malignant lesions of the esophagus were analyzed by microarrays. Gene expression profiles were compared between biopsy and surgical samples. Artificially induced epithelial-mesenchymal transition (aiEMT) was found in the surgical samples, and also occurred in mouse esophageal epithelial cell layers under an ischemic condition. Identification of clinically significant subgroups was thought to be disrupted by the disorder of the expression profile through this aiEMT.
Conclusion and Significance
This study will evoke the fundamental misinterpretation including underestimation of the prognostic evaluation power of markers by overestimation of EMT in past cancer research, and will furnish some advice for the near future as follows: 1) Understanding how long the tissues were under an ischemic condition. 2) Prevalence of biopsy samples for in vivo expression profiling with low biases on basic and clinical research. 3) Checking cancer cell contents and normal- or necrotic-tissue contamination in biopsy samples for prevalence.
Integrin-linked kinase (ILK) was first discovered as an integrin β1-subunit binding protein. It localizes at the focal adhesions and is involved in cytoskeleton remodeling. ILK overexpression and its dysregulated signaling cascades have been reported in many human cancers. Aberrant expression of ILK influenced a wide range of signaling pathways and cellular functions. Although ILK has been well characterized in many malignancies, its role in hepatocellular carcinoma (HCC) is still largely unknown.
Quantitative PCR analysis was used to examine ILK mRNA expression in HCC clinical samples. It was shown that ILK was overexpressed in 36.9% (21/57) of HCC tissues when compared to the corresponding non-tumorous livers. The overall ILK expression level was significantly higher in tumorous tissues (P = 0.004), with a significant stepwise increase in expression level along tumor progression from tumor stage I to IV (P = 0.045). ILK knockdown stable clones were established in two HCC cell lines, BEL7402 and HLE, and were subjected to different functional assays. Knockdown of ILK significantly suppressed HCC cell growth, motility and invasion in vitro and inhibited tumorigenicity in vivo. Western blot analysis revealed a reduced phosphorylated-Akt (pAkt) at Serine-473 expression in ILK knockdown stable clones when compared to control clones.
This study provides evidence about the clinical relevance of ILK in hepatocarcinogenesis. ILK was found to be progressively elevated along HCC progression. Here our findings also provide the first validation about the oncogenic capacity of ILK in vivo by suppressing its expression in HCC cells. The oncogenic role of ILK is implicated to be mediated by Akt pathway.
Over-activation of TGFβ signaling pathway and uncontrolled cell proliferation of hepatic stellate cells (HSCs) play pivotal roles in liver fibrogenesis, while the protein serine/threonine phosphatase PP2Cα was reported to negatively regulate TGFβ signaling pathway and cell cycle. Our study aimed to investigate the role of PP2Cα in liver fibrogenesis.
The effects of PP2Cα activation on liver fibrosis were investigated in human HSCs and primary rat HSCs in vitro using western blotting, real-time PCR, nuclear translocation, cell viability and cell cycle analyses. The antifibrogenic effects in carbon tetrachloride (CCl4)- and bile duct ligation (BDL)-induced mice in vivo were assessed using biochemical, histological and immunohistochemical analyses. The results demonstrated that activation of PP2Cα by overexpression or the new discovered small molecular activator NPLC0393 terminated TGFβ-Smad3 and TGFβ-p38 signaling pathways, induced cell cycle arrest in HSCs and decreased α-smooth muscle actin (α-SMA) expression, collagen deposition and hepatic hydroxyproline (HYP) level in CCl4- and BDL-induced mice.
Our findings suggested that PP2Cα activation might be an attractive new strategy for treating liver fibrosis while the small molecular activator NPLC0393 might represent a lead compound for antifibrogenic drug development. Moreover, our study might provide the first evidence for the role of PP2C family members in the fibrotic disease.