Epigenetic alterations and microRNA (miRNA) deregulation are common in hepatocellular carcinoma (HCC). The histone H3 lysine 27 (H3K27) tri‐methylating enzyme, enhancer of zeste homolog 2 (EZH2) ...mediates epigenetic silencing of gene expression and is frequently up‐regulated in human cancers. In this study we aimed to delineate the implications of EZH2 up‐regulation in miRNA deregulation and HCC metastasis. Expressions of a total of 90 epigenetic regulators were first determined in 38 pairs of primary HCCs and their corresponding nontumorous livers. We identified EZH2 and its associated polycomb repressive complex 2 (PRC2) as one of the most significantly deregulated epigenetic regulators in primary HCC samples. Up‐regulation of EZH2 was next confirmed in 69.5% (41/59) of primary HCCs. Clinicopathologically, EZH2 up‐regulation was associated with HCC progression and multiple HCC metastatic features, including venous invasion (P = 0.043), direct liver invasion (P = 0.014), and absence of tumor encapsulation (P = 0.043). We further demonstrated that knockdown of EZH2 in HCC cell lines reduced the global levels of tri‐methylated H3K27, and suppressed HCC motility in vitro and pulmonary metastasis in a nude mouse model. By interrogating the miRNA expression profile in EZH2‐knockdown cell lines and primary HCC samples, we identified a subset of miRNA that was epigenetically suppressed by EZH2 in human HCC. These included well‐characterized tumor‐suppressor miRNAs, such as miR‐139‐5p, miR‐125b, miR‐101, let‐7c, and miR‐200b. Pathway enrichment analysis revealed a common regulatory role of these EZH2‐silenced miRNAs in modulating cell motility and metastasis‐related pathways. Our findings suggest that EZH2 exerts its prometastatic function by way of epigenetic silencing of multiple tumor suppressor miRNAs. Conclusion: Our study demonstrated that EZH2 epigenetically silenced multiple miRNAs that negatively regulate HCC metastasis. (HEPATOLOGY 2012)
MicroRNAs (miRNAs) are small, noncoding RNAs that can act as oncogenes or tumor suppressors in human cancer. Our previous study showed that miR‐125b was a prognostic indicator for patients with ...hepatocellular carcinoma (HCC), but its functions and exact mechanisms in hepatic carcinogenesis are still unknown. Here we demonstrate that miR‐125b suppressed HCC cell growth in vitro and in vivo. Moreover, miR‐125b increased p21Cip1/Waf1 expression and arrested cell cycle at G1 to S transition. In addition, miR‐125b inhibited HCC cell migration and invasion. Further studies revealed that LIN28B was a downstream target of miR‐125b in HCC cells as miR‐125b bound directly to the 3′ untranslated region of LIN28B, thus reducing both the messenger RNA and protein levels of LIN28B. Silencing of LIN28B recapitulated the effects of miR‐125b overexpression, whereas enforced expression of LIN28B reversed the suppressive effects of miR‐125b. Conclusion: These findings indicate that miR‐125b exerts tumor‐suppressive effects in hepatic carcinogenesis through the suppression of oncogene LIN28B expression and suggest a therapeutic application of miR‐125b in HCC. (HEPATOLOGY 2010)
Hepatocellular carcinoma (HCC) is a prevalent cancer with an extremely high mortality rate attributed to HCC metastasis, which is the major cause of tumor recurrence and organ failure. Presence of ...tumor thrombi in the portal veins (venous metastases) is a clinicopathological feature of metastatic HCCs. In this study, we analyzed the microRNA (miRNA) expression profiles of nontumorous livers, primary HCCs, and venous metastases in the same livers from 20 HCC patients by way of TaqMan low‐density array (TLDA) and identified the precise alterations of miRNA expression from nontumorous livers to primary HCCs and venous metastases globally. By unsupervised clustering analysis, nontumorous livers were distinctly segregated from primary HCCs and venous metastases, whereas no discernible difference in the expression pattern could be found between primary HCCs and venous metastases. However, a marked global reduction of miRNA expression levels was detected in venous metastases, as compared with primary HCCs. These data suggest that miRNA deregulation is an early event in liver carcinogenesis and the later global miRNA down‐regulation aggravates the preexisting miRNA deregulation to further promote HCC metastasis. Conclusion: Our study has enriched the current understanding of the deregulation of miRNAs in HCC progression and highlighted the sequential and distinctive alterations of miRNA expression in primary HCC and venous metastasis formation. (HEPATOLOGY 2012;)
Hepatocellular carcinoma (HCC) is an aggressive tumor, with a high mortality rate due to late symptom presentation and frequent tumor recurrences and metastasis. It is also a rapidly growing tumor ...supported by different metabolic mechanisms; nevertheless, the biological and molecular mechanisms involved in the metabolic reprogramming in HCC are unclear. In this study, we found that pyruvate kinase M2 (PKM2) was frequently over-expressed in human HCCs and its over-expression was associated with aggressive clinicopathological features and poor prognosis of HCC patients. Furthermore, knockdown of PKM2 suppressed aerobic glycolysis and cell proliferation in HCC cell lines in vitro. Importantly, knockdown of PKM2 hampered HCC growth in both subcutaneous injection and orthotopic liver implantation models, and reduced lung metastasis in vivo. Of significance, PKM2 over-expression in human HCCs was associated with a down-regulation of a liver-specific microRNA, miR-122. We further showed that miR-122 interacted with the 3UTR of the PKM2 gene. Re-expression of miR-122 in HCC cell lines reduced PKM2 expression, decreased glucose uptake in vitro, and suppressed HCC tumor growth in vivo. Our clinical data and functional studies have revealed a novel biological mechanism involved in HCC metabolic reprogramming.
Hepatocellular carcinoma (HCC) is characteristically one of the most rapidly proliferating tumors which outgrows functional blood supply and results in regional oxygen deprivation. Overexpression of ...PIM1, a serine/threonine kinase, has been identified recently in human cancers. Knowledge on PIM1 in HCC is however, scarce. By immunohistochemical analysis on 56 human primary HCC samples, we observed overexpression of PIM1 in 39% of the cases. In two independent cohorts of paired primary and extra-hepatic metastatic HCC tissues, PIM1 expression was higher (p=0.002) in the extra-hepatic metastatic HCC tissues as compared with the corresponding primary HCCs. PIM1 was markedly up-regulated in multiple HCC cell lines in hypoxic condition (1% O2) versus normoxia (20% O2). Silencing of PIM1 suppressed HCC cell invasion in vitro as compared to non-target control, and decreased HCC cell proliferation in vitro and tumor growth and metastatic potential in vivo. Knockdown of PIM1 significantly reduced glucose uptake by HCC cells and was associated with decreased levels of p-AKT and key molecules in the glycolytic pathway. Taken together, PIM1 is up-regulated by hypoxia in HCC and promotes tumor growth and metastasis through facilitating cancer cell glycolysis. Targeting PIM1 may have potential role in the management of HCC.
MicroRNAs (miRNAs), an important class of small non-coding RNAs, regulate gene expression at the post-transcriptional level. miRNAs are involved in a wide range of biological processes and implicated ...in different diseases, including cancers. In this study, miRNA profiling and qRT-PCR validation revealed that miR-142-3p and miR-142-5p were significantly downregulated in hepatocellular carcinoma (HCC) and their expression levels decreased as the disease progressed. The ectopic expression of miR-142 significantly reduced HCC cell migration and invasion. Overexpression of either miR-142-3p or miR-142-5p suppressed HCC cell migration, and overexpression of both synergistically inhibited cell migration, which indicated that miR-142-3p and miR-142-5p may cooperatively regulate cell movement. miR-142-3p and miR-142-5p, which are mature miRNAs derived from the 3′- and 5′-strands of the precursor miR-142, target distinct pools of genes because of their different seed sequences. Pathway enrichment analysis showed a strong association of the putative gene targets of miR-142-3p and miR-142-5p with several cell motility-associated pathways, including those regulating actin cytoskeleton, adherens junctions, and focal adhesion. Importantly, a number of the putative gene targets were also significantly upregulated in human HCC cells. Moreover, overexpression of miR-142 significantly abrogated stress fiber formation in HCC cells and led to cell shrinkage. This study shows that mature miR-142 pairs collaboratively regulate different components of distinct signaling cascades and therefore affects the motility of HCC cells.
Introduction: Treatment decisions based on patient-specific molecular features are central to personalized cancer precision medicine. Oftentimes treatment response of an individual patient remains an ...issue of trial and error. Expression profiling has been utilized to study molecular subtypes of tumor entities and their impact on treatment outcome. Biological effector programs, such as cellular senescence, however, remain largely understudied. Syngeneic mouse models of cancer that can reproduce critical molecular features of human malignancies could serve as useful models to explore genetic determinants of drug sensitivity, and, likewise, to unveil molecular mechanisms of treatment resistance. Here, we focus on the involvement of therapy-induced senescence on treatment outcome in mouse models and patients diagnosed with diffuse large B-cell lymphoma (DLBCL).
Methods: We present and characterize here the utilization of Eµ-myc transgenic lymphomas as a faithful model of chemoresistance and demonstrate its cross-species validity for DLBCL patients. Specifically, primary Eµ-myc lymphomas, of which we generated gene expression profiles (GEP) at diagnosis, were exposed to genotoxic therapy in vivo, and subsequently monitored regarding long-term outcome in a clinical trial-like design. Lymphoma senescence capability, a central drug effector principle, was studied in mice by unbiased approaches as well as loss- and gain-of-function genetics.
Results: Investigation of DLBCL-established gene expression based subtypes related to cell-of-origin (COO - i.e. GCB/ABC subtypes) and distinct DLBCL biologies (e.g. comprehensive consensus clusters CCC) using machine-learning methods demonstrated their relevance in the murine platform. Moreover, our findings show an important role of histone H3 lysine 9-trimethylation (H3K9me3) for senescence induction and treatment outcome as demonstrated by shorter time to death and time to relapse of mice bearing lymphomas with engineered loss of the H3K9me3-critical methyltransferase Suv39h1 on one hand and lymphomas with genetically transferred or endogenous overexpression of H3K9-active demethylases on the other hand. Furthermore, expression levels of H3K9me3-specific demethylases stratified unmodified Eµ-myc lymphomas and DLBCL patients into two groups with superior outcome for those with lower levels. In line with these findings, DLBCL patients with high levels of the senescence-associated H3K9me3 mark in their lymphomas presented with significantly longer survival times. Further transcriptomics-based investigations of our clinical-trial like mouse model and DLBCL patients suggests the presence of a molecular network distinguishing lymphomas into a clinically superior senescence responder from an inferior non-responder group.
Conclusions: Our results conclude that Eµ-myc transgenic lymphomas serve as faithful model for human DLBCL and the importance of therapy-induced senescence for treatment outcome of DLBCL patients. Our data suggest the integration of tractable, transgenic mouse models to the repertoire of functional test platforms to better implement lesion- and state-based decisions in personalized cancer precision medicine. Ongoing mouse model-based work aims at specific targeting of aberrant demethylase activities and synthetic lethal approaches to selectively eliminate potentially detrimental senescent lymphoma cells after chemotherapy to assess therapeutic long-term effects and to determine the conditions for future early-phase DLBCL clinical testing.
No relevant conflicts of interest to declare.
Introduction: Acute myeloid leukemia (AML) is a heterogeneous disease, for which biologically grounded predictors of outcome remain an urgent clinical need. In addition to tumor cell death, ...antineoplastic drugs have been shown to mediate a long-lasting growth arrest of vital, metabolically active tumor cells termed therapy-induced senescence (TIS) in various cancer entities and model systems, but predictive investigations on TIS are lacking. We hypothesized here that an ex vivo assessment of TIS in blast samples from AML patients at diagnosis might serve as a biomarker of outcome.
Methods: We established assays to quantitatively detect TIS in a primary culture setting of purified primary human AML specimens (n > 30) assessed through cytochemical and fluorescence-based analysis of senescence-associated β-galactosidase (SA-β-gal) activity and other senescence markers (such as p16INK4a, H3K9me3) in concert with downregulation of proliferation markers (Ki67, EdU). AML samples were subjected to standard karyotype and molecular genetics analyses, defining risk groups according to the European Leukemia Net (ELN) genetic risk stratification.
Results: We found the individual fractions of senescent AML blasts in response to the standard anti-leukemic agents Daunorubicin and Cytarabine samples to be positively correlated with better disease-free (p= 0.042) and overall survival (p= 0.004) of the patients. Furthermore, a more favorable molecular risk group (p= 0.005), normal karyotype (p= 0.007), and NPM1 mutations (p= 0.018) as well as DNMT3A mutations (p < 0.001) were associated with higher TIS levels in our ex vivo senescence assay.
Discussion: Our study is the first report providing direct evidence that cellular senescence induced in patient-derived AML blasts by chemotherapeutic drugs ex vivo serves as a predictor of patient long-term responsiveness to standard induction therapy. TIS might explain the underlying biology of current para-clinical risk indicators, and also seems to identify subgroups of superior outcome in pre-defined risk groups including patients with DNMT3A-mutant disease. Additional investigations exploring senescence in response to other AML-relevant agents and functional analyses expanding to patient-derived xenograft (PDX) AML models will be presented at the meeting.
No relevant conflicts of interest to declare.
PDF
