A lactate-induced response to hypoxia.

Organisms must be able to respond to low oxygen in a number of homeostatic and pathological contexts. Regulation of hypoxic responses via the hypoxia-inducible factor (HIF) is well established, but evidence indicates that other, HIF-independent mechanisms are also involved. Here, we report a hypoxic response that depends on the accumulation of lactate, a metabolite whose production increases in hypoxic conditions. We find that the NDRG3 protein is degraded in a PHD2/VHL-dependent manner in normoxia but is protected from destruction by binding to lactate that accumulates under hypoxia. The stabilized NDRG3 protein binds c-Raf to mediate hypoxia-induced activation of Raf-ERK pathway, promoting angiogenesis and cell growth. Inhibiting cellular lactate production abolishes the NDRG3-mediated hypoxia responses. Our study, therefore, elucidates the molecular basis for lactate-induced hypoxia signaling, which can be exploited for the development of therapies targeting hypoxia-induced diseases.

Regulation of hypoxia responses by flavin adenine dinucleotide-dependent modulation of HIF-1α protein stability.

Oxygen deprivation induces a range of cellular adaptive responses that enable to drive cancer progression. Here, we report that lysine-specific demethylase 1 (LSD1) upregulates hypoxia responses by demethylating RACK1 protein, a component of hypoxia-inducible factor (HIF) ubiquitination machinery, and consequently suppressing the oxygen-independent degradation of HIF-1α. This ability of LSD1 is attenuated during prolonged hypoxia, with a decrease in the cellular level of flavin adenine dinucleotide (FAD), a metabolic cofactor of LSD1, causing HIF-1α downregulation in later stages of hypoxia. Exogenously provided FAD restores HIF-1α stability, indicating a rate-limiting role for FAD in LSD1-mediated HIF-1α regulation. Transcriptomic analyses of patient tissues show that the HIF-1 signature is highly correlated with the expression of LSD1 target genes as well as the enzymes of FAD biosynthetic pathway in triple-negative breast cancers, reflecting the significance of FAD-dependent LSD1 activity in cancer progression. Together, our findings provide a new insight into HIF-mediated hypoxia response regulation by coupling the FAD dependence of LSD1 activity to the regulation of HIF-1α stability.

LAD1 expression is associated with the metastatic potential of colorectal cancer cells.

BACKGROUND: Anchoring filament protein ladinin-1 (LAD1) was related to the aggressive progression of breast, lung, laryngeal and thyroid cancers. However, the association of LAD1 with colorectal cancer remained unknown. Here, to determine the relationship of LAD1 with colorectal cancer progression, we explored the effect of LAD1 loss on the malignant features of colorectal cancer cells. METHODS: We constructed LAD1-depleted cell lines and examined the effect of LAD1 deficiency on the phenotypic and molecular features of colorectal cancer cells in vitro. The function of LAD1 in metastasis in vivo was examined by establishing a spleen-to-liver metastasis mouse model. LAD1 protein expression in colorectal cancer patient specimens was assessed by immunohistochemistry of tumor microarrays. RESULTS: We found that LAD1 was abundant in most colorectal cancer cells. In addition, high expression of LAD1 significantly correlated with poor patient outcome. LAD1 depletion inhibited the migration and invasion of two different colorectal cancer cell lines, SW620 and Caco-2, without affecting their proliferation. In addition, LAD1 loss led to defects in liver metastasis of SW620 cells in the mouse model. Immunohistochemistry of colorectal cancer tissues revealed LAD1 enrichment in metastatic tissues compared to that in primary tumor and normal tissues. CONCLUSION: These results suggest that LAD1 expression is associated with the metastatic progression of colorectal cancer by promoting the migration and invasion of cancer cells.

Recurrence-associated pathways in hepatitis B virus-positive hepatocellular carcinoma.

BACKGROUND: Despite the recent identification of several prognostic gene signatures, the lack of common genes among experimental cohorts has posed a considerable challenge in uncovering the molecular basis underlying hepatocellular carcinoma (HCC) recurrence for application in clinical purposes. To overcome the limitations of individual gene-based analysis, we applied a pathway-based approach for analysis of HCC recurrence. RESULTS: By implementing a permutation-based semi-supervised principal component analysis algorithm using the optimal principal component, we selected sixty-four pathways associated with hepatitis B virus (HBV)-positive HCC recurrence (p < 0.01), from our microarray dataset composed of 142 HBV-positive HCCs. In relation to the public HBV- and public hepatitis C virus (HCV)-positive HCC datasets, we detected 46 (71.9%) and 18 (28.1%) common recurrence-associated pathways, respectively. However, overlap of recurrence-associated genes between datasets was rare, further supporting the utility of the pathway-based approach for recurrence analysis between different HCC datasets. Non-supervised clustering of the 64 recurrence-associated pathways facilitated the classification of HCC patients into high- and low-risk subgroups, based on risk of recurrence (p < 0.0001). The pathways identified were additionally successfully applied to discriminate subgroups depending on recurrence risk within the public HCC datasets. Through multivariate analysis, these recurrence-associated pathways were identified as an independent prognostic factor (p < 0.0001) along with tumor number, tumor size and Edmondson's grade. Moreover, the pathway-based approach had a clinical advantage in terms of discriminating the high-risk subgroup (N = 12) among patients (N = 26) with small HCC (<3 cm). CONCLUSIONS: Using pathway-based analysis, we successfully identified the pathways involved in recurrence of HBV-positive HCC that may be effectively used as prognostic markers.

SH3RF2 functions as an oncogene by mediating PAK4 protein stability.

SH3RF (SH3-domain-containing RING finger protein) family members, SH3RF1-3, are multidomain scaffold proteins involved in promoting cell survival and apoptosis. In this report, we show that SH3RF2 is an oncogene product that is overexpressed in human cancers and regulates p21-activated kinase 4 (PAK4) protein stability. Immunohistochemical analysis of 159 colon cancer tissues showed that SH3RF2 expression levels are frequently elevated in cancer tissues and significantly correlate with poor prognostic indicators, including increased invasion, early recurrence and poor survival rates. We also demonstrated that PAK4 protein is degraded by the ubiquitin-proteasome system and that SH3RF2 inhibits PAK4 ubiquitination via physical interaction-mediated steric hindrance, which results in the upregulation of PAK4 protein. Moreover, ablation of SH3RF2 expression attenuates TRADD (TNFR-associated death domain) recruitment to tumor necrosis factor-α (TNF-α) receptor 1 and hinders downstream signals, thereby inhibiting NF-κB (nuclear factor-kappaB) activity and enhancing caspase-8 activity, in the context of TNF-α treatment. Notably, ectopic expression of SH3RF2 effectively prevents apoptosis in cancer cells and enhances cell migration, colony formation and tumor growth in vivo. Taken together, our results suggest that SH3RF2 is an oncogene that may be a definitive regulator of PAK4. Therefore, SH3RF2 may represent an effective therapeutic target for cancer treatment.

Integrated analysis of prognostic gene expression profiles from hepatitis B virus-positive hepatocellular carcinoma and adjacent liver tissue.

BACKGROUND: The tissue environment in the region of hepatocellular carcinoma (HCC) influences both vascular invasion and recurrence. Thus, HCC patient prognosis depends on the characteristics not only of the tumor but also those of adjacent surrounding liver tissue. MATERIALS AND METHODS: Expression profiles of both tumor and adjacent liver tissue following curative resection were measured to discriminate 56 hepatitis B virus-positive HCC patients into subgroups based on survival risk. This approach was further tested in 40 patients. RESULTS: Expression profiles of both tumor and adjacent liver tissue successfully discriminated 56 training samples into 2 subgroups, those at low- or high-risk for survival and recurrence. However, the prognostic gene set selected for tumor tissue was quite different from that for adjacent tissues. This variation in prognostic genes resulted in a change in allocation of patients within each low- or high-risk group. Combination of survival subgroups from tumor and adjacent liver tissue significantly improved the prediction of prognostic outcome. This integrative approach was confirmed to be effective in a further 40 test patients. A clinicopathological study showed that survival subgroups divided by tumor and adjacent liver tissue gene expression were also statistically associated with UICC stage and extent of cell differentiation, respectively. CONCLUSIONS: Variation in gene expression during the nontumor stage as well as the tumor stage may affect the prognosis of HCC patients, and integration of the gene expression profiles of HCC and adjacent liver tissue increases discriminatory effectiveness between patient groups, predicting clinical outcomes with enhanced statistical reliability.

Functional switching of TGF-beta1 signaling in liver cancer via epigenetic modulation of a single CpG site in TTP promoter.

BACKGROUND & AIMS: Acquisition of resistance to the antiproliferative effect of transforming growth factor (TGF)-beta1 is crucial for the malignant progression of cancers. In this study, we sought to determine whether deregulated expression of tristetrapolin (TTP), a negative posttranscriptional regulator of c-Myc, confers resistance to the antiproliferative effects of TGF-beta1 on liver cancer cells. METHODS: The epigenetics of TTP promoter regulation and its effects on TGF-beta1 signaling were examined in hepatocellular carcinoma (HCC) cell lines and patient tissues. RESULTS: TTP was down-regulated in HCC cell lines (10/11), compared with normal liver, as well as in tumor tissues (19/24) from paired HCC specimens. Methylation of a specific single CpG site located within the TGF-beta1-responsive region (TRR) of the TTP promoter was significantly associated with TTP down-regulation in both HCC cell lines and tumor tissues (r = -0.606383, P < .001). The singly methylated CpG site was specifically bound by a transcriptional repressor complex consisting of MECP2/c-Ski/DNMT3A and abolished the TGF-beta1-induced as well as basal-level expression of TTP. The epigenetic inactivation of TTP led to an increased half-life of c-Myc mRNA and blocked the cytostatic effect of TGF-beta1. Statistically significant correlations were observed between the single CpG site methylation and expression levels of TTP or c-Myc in clinical samples of HCC. CONCLUSIONS: Abrogation of the post-transcriptional regulation of c-Myc via methylation of a specific single CpG site in the TTP promoter presents a novel mechanism for the gain of selective resistance to the antiproliferative signaling of TGF-beta1 in HCC.

CIIA induces the epithelial-mesenchymal transition and cell invasion.

Epithelial-mesenchymal transition (EMT) and the acquisition of invasive potential are key events in tumor progression. We now show that CIIA, originally identified as an anti-apoptotic protein, induces the EMT and promotes cell migration and invasion. Ectopic expression of CIIA induced down-regulation of E-cadherin and claudin-1 as well as up-regulation of N-cadherin in MDCK cells. It also disrupted the differentiated epithelial morphology of MDCK cells grown in three-dimensional Matrigel cultures as well as increased the migration and invasion of MDCK cells in vitro. Furthermore, depletion of endogenous CIIA by RNA interference inhibited the migration and invasion of HeLa cells, and this inhibition was abolished by RNA interference-mediated depletion of claudin-1. These results suggest that CIIA functions as an inducer of cell invasion, and this effect is mediated, at least in part, through down-regulation of claudin-1.

Gene expression profiling in mouse liver infected with Clonorchis sinensis metacercariae.

Clonorchis sinensis, the parasite that causes clonorchiasis, is endemic in many Asian countries, and infection with the organism drives changes in the liver tissues of the host. However, information regarding the molecular events in clonorchiasis remains limited, and little is currently known about host-pathogen interactions in clonorchiasis. In this study, we assessed the gene expression profiles in mice livers via DNA microarray analysis 1, 2, 4, and 6 weeks after induced metacercariae infection. Functional clustering of the gene expression profile showed that the immunity-involved genes were induced in the livers of the mice at the early stage of metacercariae infection, whereas immune responses were reduced in the 6-week liver tissues after infection in which the metacercariae became adult flukes. Many genes involved in fatty acid metabolism, including Peci, Cyp4a10, Acat1, Ehhadh, Gcdh, and Cyp2 family were downregulated in the infected livers. On the other hand, the liver tissues infected with the parasite expressed Wnt signaling molecules such as Wnt7b, Fzd6, and Pdgfrb and cell cycle-regulating genes including cyclin-D1, Cdca3, and Bcl3. These investigations constitute an excellent starting point for increased understanding of the molecular mechanisms underlying host-pathogen interaction during the development of C. sinensis in the host liver.

Lysyl oxidase like 4, a novel target gene of TGF-beta1 signaling, can negatively regulate TGF-beta1-induced cell motility in PLC/PRF/5 hepatoma cells.

Transforming growth factor-beta1 (TGF-beta1) is a multi-functional cytokine involved in the regulation of cell proliferation, differentiation and extracellular matrix formation. In search for novel genes mediating the TGF-beta1 function at downstream signaling, we performed a cDNA microarray analysis and identified 60 genes whose expression is regulated by TGF-beta1 in the liver cancer cell line PLC/PRF/5. Among them, we report here lysyl oxidase like 4 (LOXL4) as a novel target of TGF-beta1 signaling, and provide experimental evidence for its expression regulation and function. LOXL4 was found to be the only member of LOX family whose expression is induced by TGF-beta1 in hepatoma cells. Deletion mapping of the LOXL4 promoter indicated that the TGF-beta1 regulation of LOXL4 expression is mediated through the binding of AP1 transcription factor to a conserved region of the promoter. This was confirmed by the chromatin immunoprecipitation assay that captured c-Fos-bound chromatin from TGF-beta1-treated cells. Forced expression of LOXL4 in PLC/PRF/5 cells resulted in inhibition of cell motility through Matrigel in the presence of TGF-beta1 treatment. In parallel, LOXL4 suppressed the expression of laminins and alpha3 integrin and the activity of MMP2. These results suggest that LOXL4 may function as a negative feedback regulator of TGF-beta1 in cell invasion by inhibiting the metabolism of extracellular matrix (ECM) components.

RNF25 promotes gefitinib resistance in EGFR-mutant NSCLC cells by inducing NF-κB-mediated ERK reactivation.

Non-small cell lung cancer (NSCLC) patients with EGFR mutations initially respond well to EGFR tyrosine kinase inhibitors (TKIs) but eventually exhibit acquired or innate resistance to the therapies typically due to gene mutations, such as EGFR T790M mutation or a second mutation in the downstream pathways of EGFR. Importantly, a significant portion of NSCLC patients shows TKI resistance without any known mechanisms, calling more comprehensive studies to reveal the underlying mechanisms. Here, we investigated a synthetic lethality with gefitinib using a genome-wide RNAi screen in TKI-resistant EGFR-mutant NSCLC cells, and identified RNF25 as a novel factor related to gefitinib resistance. Depletion of RNF25 expression substantially sensitized NSCLC cells to gefitinib treatment, while forced expression of RNF25 augmented gefitinib resistance in sensitive cells. We demonstrated that RNF25 mediates NF-κB activation in gefitinib-treated cells, which, in turn, induces reactivation of ERK signal to cause the drug resistance. We identified that the ERK reactivation occurs via the function of cytokines, such as IL-6, whose expression is transcriptionally induced in a gefitinib-dependent manner by RNF25-mediated NF-κB signals. These results suggest that RNF25 plays an essential role in gefitinib resistance of NSCLC by mediating cross-talk between NF-κB and ERK pathways, and provide a novel target for the combination therapy to overcome TKI resistance of NSCLC.

Novel candidate targets of Wnt/beta-catenin signaling in hepatoma cells.

The activity of beta-catenin/TCF, the key component of Wnt signaling pathway, is frequently deregulated in HCC, resulting in the activation of genes whose dysregulation has significant consequences on tumor development. Therefore, identifying the target genes of Wnt signaling is important for understanding beta-catenin-mediated carcinogenesis. We analyzed the transcriptome profile of human hepatoma cell lines using cDNA microarrays representing 15,127 unique, liver-enriched gene loci to identify the target genes of beta-catenin-mediated transcription (p<0.005). This analysis yielded 130 potential Wnt-associated classifier genes, and we found 33 of them contain consensus TCF-binding sites in presumptive transcriptional regulatory sequences. These genes were, then, tested for their Wnt-dependence of expression in experimental models of Wnt activation. Genes such as RPL29, NEDD4L, FUT8, LYZ, STMN2, STARD7 and KIAA0998 were proven to be up-regulated upon Wnt/beta-catenin activation. Gene ontology analysis of the 33 candidate genes indicated the presence of functional categories relevant to Wnt pathway such as cell growth, proliferation, adhesion and signal transduction. In conclusion, we identified a number of candidate Wnt/beta-catenin target genes that can be useful for studying the role of altered Wnt signaling in liver cancer development, and showed that some of them might be direct targets of Wnt signaling in hepatoma cells.

AKT-induced PKM2 phosphorylation signals for IGF-1-stimulated cancer cell growth.

Pyruvate kinase muscle type 2 (PKM2) exhibits post-translational modifications in response to various signals from the tumor microenvironment. Insulin-like growth factor 1 (IGF-1) is a crucial signal in the tumor microenvironment that promotes cell growth and survival in many human cancers. Herein, we report that AKT directly interacts with PKM2 and phosphorylates it at Ser-202, which is essential for the nuclear translocation of PKM2 protein under stimulation of IGF-1. In the nucleus, PKM2 binds to STAT5A and induces IGF-1-stimulated cyclin D1 expression, suggesting that PKM2 acts as an important factor inducing STAT5A activation under IGF-1 signaling. Concordantly, overexpression of STAT5A in cells deficient in PKM2 expression failed to restore IGF-induced growth, whereas reconstitution of PKM2 in PKM2 knockdown cells restored the IGF-induced growth capacity. Our findings suggest a novel role of PKM2 in promoting the growth of cancers with dysregulated IGF/phosphoinositide 3-kinase/AKT signaling.

A basal-like breast cancer-specific role for SRF-IL6 in YAP-induced cancer stemness.

The switch between stem/progenitor cell expansion and differentiation is critical for organ homeostasis. The mammalian Hippo pathway effector and oncoprotein YAP expands undifferentiated stem/progenitor cells in various tissues. However, the YAP-associated transcription factors and downstream targets underlying this stemness-promoting activity are poorly understood. Here we show that the SRF-IL6 axis is the critical mediator of YAP-induced stemness in mammary epithelial cells and breast cancer. Specifically, serum response factor (SRF)-mediated binding and recruitment of YAP to mammary stem cell (MaSC) signature-gene promoters induce numerous MaSC signature genes, among which the target interleukin (IL)-6 is critical for YAP-induced stemness. High SRF-YAP/TAZ expression is correlated with IL6-enriched MaSC/basal-like breast cancer (BLBC). Finally, we show that this high SRF expression enables YAP to more efficiently induce IL6 and stemness in BLBC compared with luminal-type breast cancer. Collectively, our results establish the importance of SRF-YAP-IL6 signalling in promoting MaSC-like properties in a BLBC-specific manner.

Beta-lapachone, a modulator of NAD metabolism, prevents health declines in aged mice.

NADH-quinone oxidoreductase 1 (NQO1) modulates cellular NAD(+)/NADH ratio which has been associated with the aging and anti-aging mechanisms of calorie restriction (CR). Here, we demonstrate that the facilitation of NQO1 activity by feeding ß-lapachone (ßL), an exogenous NQO1 co-substrate, prevented age-dependent decline of motor and cognitive function in aged mice. ßL-fed mice did not alter their food-intake or locomotor activity but did increase their energy expenditure as measured by oxygen consumption and heat generation. Mitochondrial structure and numbers were disorganized and decreased in the muscles of control diet group but those defects were less severe in ßL-fed aged mice. Furthermore, for a subset of genes associated with energy metabolism, mice fed the ßL-diet showed similar changes in gene expression to the CR group (fed 70% of the control diet). These results support the potentiation of NQO1 activity by a ßL diet and could be an option for preventing age-related decline of muscle and brain functions.

Differential modulatory effects of rosiglitazone and pioglitazone on white adipose tissue in db/db mice.

AIMS: this study was performed to clarify the different action mechanisms through which rosiglitazone and pioglitazone regulate lipogenesis in white adipose tissues of db/db mice, an animal model of diabetes. MAIN METHODS: male C57BLKS/J-Lepr(db/db) (db/db) mice were used for all experiments. Rosiglitazone or pioglitazone were administered once daily by oral gavage for 4 weeks at concentrations of 20mg/kg and 75 mg/kg, respectively. At 0, 3, 6, 9, 12, 15, 21, and 28 days of administration, body weights and blood glucose were determined. At the end of experiment, adiposity and gene expression were confirmed by perilipin A immunostaining and real-time PCR. KEY FINDINGS: pioglitazone treatment increased fat mass and the surface area of adipocytes more than rosiglitazone at dosages with equivalent effects on plasma glucose. Lipid parameters including plasma total cholesterol and triglycerides were decreased more in rosiglitazone-treated mice. Relative mRNA expression levels for lipid synthesis and transport including diacylglycerol acyltransferase (DGAT1/2), fatty acid translocase (CD36/FAT), fatty acid transport protein (FATP) were increased in pioglitazone-treated group compared to rosiglitazone-treated mice, but mRNA expression levels of ß-oxidation-related genes acyl-Coenzyme A dehydrogenase, very long chain (Acadvl), acyl-Coenzyme A dehydrogenase, medium chain (Acadm), and the energy expenditure-related genes triosephosphate isomerase 1 (Tpi1) and carnitine palmitoyltransferase 1b (Cpt1b) were decreased. SIGNIFICANCE: these results suggest that pioglitazone activates lipid deposition by increasing lipid synthesis and transport, but rosiglitazone stimulates ß-oxidation and energy expenditure in adipocytes of db/db mice.

Gene expression analysis of terminal differentiation of human melanoma cells highlights global reductions in cell cycle-associated genes.

Defects in differentiation are frequently observed in cancer cells. By appropriate treatment specific tumor cell types can be induced to terminally differentiate. Metastatic HO-1 human melanoma cells treated with IFN-beta plus mezerein (MEZ) undergo irreversible growth arrest and terminal differentiation followed by apoptosis. In order to define the molecular changes associated with this process, changes in gene expression were analyzed by cDNA microarray hybridization and by semi-quantitative and quantitative RT-PCRs of representative 44 genes. The expression of 210 genes was changed more than two-fold at either 8 or 24 h post-treatment (166 up and 44 down). Major biological processes associated with the up-regulated genes were response to endogenous/exogenous stimuli (38%), cell proliferation (13%), cell death (16%) and development (30%). Approximately 34% of the down-regulated genes were associated with cell cycle, 9% in DNA replication and 11% in chromosome organization, respectively. Suppression of cell cycle associated genes appeared to directly correlate with growth arrest observed in the terminal differentiation process. Expression of Calpain 3 (CAPN3) variant 6 was suppressed by the combined treatment and maintained high in various melanoma cell lines. However, over-expression of the CAPN3 did not significantly affect growth kinetics and cell viability, suggesting that up-regulation of CAPN3 alone may not be a causative, but an associated change with melanoma development. This analysis provides further insights into the spectrum of up-regulated and the first detailed investigation of down-regulated gene changes associated with and potentially causative of induction of loss of proliferative capacity and terminal differentiation in human melanoma cells.

Osteopontin splice variants differentially modulate the migratory activity of hepatocellular carcinoma cell lines.

Osteopontin (OPN, SPP1) is a secretory extracellular matrix protein that has been implicated in cancer-associated mechanisms such as metastasis, invasion and angiogenesis. Three OPN isoforms (OPN-a, -b and -c) derived from alternative splicing are known to exist, but their functional specificity remains unclear. Here, we found that the expression profile of OPN isoforms in hepatocellular carcinoma (HCC) cell lines and patient tissues were correlated with specific cellular phenotypes and tumorigenicity of HCC. Thus, SK-Hep1 cells with a robust migratory capacity dominantly expressed both OPN-a and -b, but non-migratory cell lines such as Hep3B and PLC/PRF/5 mainly expressed OPN-c. Moreover, tumor tissues predominantly expressed OPN-a and -b, whereas normal liver tissues mainly expressed OPN-c. Transwell infiltration and wound-induced migration assays revealed that both OPN-a and -b induced Hep3B cell migration, while OPN-c had no significant effects. By contrast, OPN-c suppressed the migratory activity of SK-Hep1 cells although no significant changes were induced by OPN-a. Consistently, OPN isoforms differentially activated migration-associated signaling pathways such that OPN-a and -b increased the expression of urokinase type plasminogen activator and the phosphorylation of p42/p44 MAP kinase, but these pathways were not activated by OPN-c. Thus, the findings of the present study suggest that OPN splice variants differentially couple to signaling pathways to modulate the migratory property of HCC cells and that this is one of the mechanisms underlying the pathological heterogeneity of HCC progression.

Hepatocellular carcinoma model cell lines with two distinct migration modes.

Migration is an essential feature of metastatic cancer cells. To understand how motility is regulated in hepatocellular carcinoma, we analyzed gene expression profiles of mouse model cell lines we established from transgenic mice carrying SV40 large T antigen. A non-motile HC9 cell line was isolated from mouse liver tumors, and two additional cell lines, HCM1 and HCM4, were derived from HC9 cells. We found that both HCM1 and HCM4 cells were substantially more migratory than HC9, and that HCM1 generated tumor nodules in nude mice. In contrast to HCM4 cells that exhibited mesenchymal cell-type gene expression similar to HC9 cells, HCM1 cells appeared to have undergone a mesenchymal-amoeboidal transition. Thus, HCM1 and HCM4 cells have distinct migration and gene expression patterns, and together with HC9 cells, they can serve as model cell lines for understanding how migration is acquired and controlled in hepatocellular carcinoma.

STMN2 is a novel target of beta-catenin/TCF-mediated transcription in human hepatoma cells.

The activity of beta-catenin/TCF, the key component of Wnt signaling pathway, is frequently deregulated in human cancers, resulting in the activation of genes whose dysregulation has significant consequences on tumor development. Therefore, identifying the target genes of Wnt signaling is important for understanding beta-catenin-mediated carcinogenesis. Here, we report STMN2, a gene implicated in the regulation of microtubule dynamics, as a novel target of beta-catenin-mediated transcription. STMN2 was up-regulated in hepatoma and cirrhotic liver tissues compared to normal liver and also in cell lines where beta-catenin/TCF is constitutively activated. Transient activation of beta-catenin/TCF either by transfection of a constitutively active form of beta-catenin or by LiCl treatment induced the STMN2 mRNA expression in PLC/PRF/5 cells. Of the four members of STMN gene family, only STMN2 showed a Wnt-dependent expression pattern. Through promoter mapping and chromatin immunoprecipitation assays, we found that STMN2 is a direct target of beta-catenin/TCF-mediated transcription and that the TCF binding site at -1713 of STMN2 promoter is critical for beta-catenin/TCF-dependent expression regulation. siRNA-mediated knock-down of STMN2 expression indicated that STMN2 is required for maintaining the anchorage-independent growth state of beta-catenin/TCF-activated hepatoma cells. Our results suggest that STMN2 might be a novel player of beta-catenin/TCF-mediated carcinogenesis in the liver.