Nuclear F-actin assembly on damaged chromatin is regulated by DYRK1A and Spir1 phosphorylation.

Nuclear actin-based movements support DNA double-strand break (DSB) repair. However, molecular determinants that promote filamentous actin (F-actin) formation on the damaged chromatin remain undefined. Here we describe the DYRK1A kinase as a nuclear activity that promotes local F-actin assembly to support DSB mobility and repair, accomplished in part by its targeting of actin nucleator spire homolog 1 (Spir1). Indeed, perturbing DYRK1A-dependent phosphorylation of S482 mis-regulated Spir1 accumulation at damaged-modified chromatin, and led to compromised DSB-associated actin polymerization and attenuated DNA repair. Our findings uncover a role of the DYRK1A-Spir1 axis in nuclear actin dynamics during early DSB responses, and highlight the intricate details of nuclear cytoskeletal network in DSB repair and genome stability maintenance.

Latent membrane protein 1 and macrophage-derived TNFα synergistically activate and mobilize invadopodia to drive invasion of nasopharyngeal carcinoma.

Invadopodia are actin-rich membrane protrusions that digest the matrix barrier during cancer metastasis. Since the discovery of invadopodia, they have been visualized as localized and dot-like structures in different types of cancer cells on top of a 2D matrix. In this investigation of Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC), a highly invasive cancer frequently accompanied by neck lymph node and distal organ metastases, we revealed a new form of invadopodium with mobilizing features. Integration of live-cell imaging and molecular assays revealed the interaction of macrophage-released TNFα and EBV-encoded latent membrane protein 1 (LMP1) in co-activating the EGFR/Src/ERK/cortactin and Cdc42/N-WASP signaling axes for mobilizing the invadopodia with lateral movements. This phenomenon endows the invadopodia with massive degradative power, visualized as a shift of focal dot-like digestion patterns on a 2D gelatin to a dendrite-like digestion pattern. Notably, single stimulation of either LMP1 or TNFα could only enhance the number of ordinary dot-like invadopodia, suggesting that the EBV infection sensitizes the NPC cells to form mobilizing invadopodia when encountering a TNFα-rich tumor microenvironment. This study unveils the interplay of EBV and stromal components in driving the invasive potential of NPC via unleashing the propulsion of invadopodia in overcoming matrix hurdles. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Regulation of chromatin architecture by the PWWP domain-containing DNA damage-responsive factor EXPAND1/MUM1.

Dynamic changes of chromatin structure facilitate diverse biological events, including DNA replication, repair, recombination, and gene transcription. Recent evidence revealed that DNA damage elicits alterations to the chromatin to facilitate proper checkpoint activation and DNA repair. Here we report the identification of the PWWP domain-containing protein EXPAND1/MUM1 as an architectural component of the chromatin, which in response to DNA damage serves as an accessory factor to promote cell survival. Depletion of EXPAND1/MUM1 or inactivation of its PWWP domain resulted in chromatin compaction. Upon DNA damage, EXPAND1/MUM1 rapidly concentrates at the vicinity of DNA damage sites via its direct interaction with 53BP1. Ablation of this interaction impaired damage-induced chromatin decondensation, which is accompanied by sustained DNA damage and hypersensitivity to genotoxic stress. Collectively, our study uncovers a chromatin-bound factor that serves an accessory role in coupling damage signaling with chromatin changes in response to DNA damage.

Physical exercise-induced hippocampal neurogenesis and antidepressant effects are mediated by the adipocyte hormone adiponectin.

Adiponectin (ADN) is an adipocyte-secreted protein with insulin-sensitizing, antidiabetic, antiinflammatory, and antiatherogenic properties. Evidence is also accumulating that ADN has neuroprotective activities, yet the underlying mechanism remains elusive. Here we show that ADN could pass through the blood-brain barrier, and elevating its levels in the brain increased cell proliferation and decreased depression-like behaviors. ADN deficiency did not reduce the basal hippocampal neurogenesis or neuronal differentiation but diminished the effectiveness of exercise in increasing hippocampal neurogenesis. Furthermore, exercise-induced reduction in depression-like behaviors was abrogated in ADN-deficient mice, and this impairment in ADN-deficient mice was accompanied by defective running-induced phosphorylation of AMP-activated protein kinase (AMPK) in the hippocampal tissue. In vitro analyses indicated that ADN itself could increase cell proliferation of both hippocampal progenitor cells and Neuro2a neuroblastoma cells. The neurogenic effects of ADN were mediated by the ADN receptor 1 (ADNR1), because siRNA targeting ADNR1, but not ADNR2, inhibited the capacity of ADN to enhance cell proliferation. These data suggest that adiponectin may play a significant role in mediating the effects of exercise on hippocampal neurogenesis and depression, possibly by activation of the ADNR1/AMPK signaling pathways, and also raise the possibility that adiponectin and its agonists may represent a promising therapeutic treatment for depression.

Differential regulation of RNF8-mediated Lys48- and Lys63-based poly-ubiquitylation.

Pairing of a given E3 ubiquitin ligase with different E2s allows synthesis of ubiquitin conjugates of different topologies. While this phenomenon contributes to functional diversity, it remains largely unknown how a single E3 ubiquitin ligase recognizes multiple E2s, and whether identical structural requirements determine their respective interactions. The E3 ubiquitin ligase RNF8 that plays a critically important role in transducing DNA damage signals, interacts with E2s UBCH8 and UBC13, and catalyzes both K48- and K63-linked ubiquitin chains. Interestingly, we report here that a single-point mutation (I405A) on the RNF8 polypeptide uncouples its ability in catalyzing K48- and K63-linked ubiquitin chain formation. Accordingly, while RNF8 interacted with E2s UBCH8 and UBC13, its I405A mutation selectively disrupted its functional interaction with UBCH8, and impaired K48-based poly-ubiquitylation reactions. In contrast, RNF8 I405A preserved its interaction with UBC13, synthesized K63-linked ubiquitin chains, and assembled BRCA1 and 53BP1 at sites of DNA breaks. Together, our data suggest that RNF8 regulates K48- and K63-linked poly-ubiquitylation via differential RING-dependent interactions with its E2s UBCH8 and UBC13, respectively.

Group I p21-activated kinases facilitate Tax-mediated transcriptional activation of the human T-cell leukemia virus type 1 long terminal repeats.

BACKGROUND: Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia and tropical spastic paraparesis. HTLV-1 encodes transactivator protein Tax that interacts with various cellular factors to modulate transcription and other biological functions. Additional cellular mediators of Tax-mediated transcriptional activation of HTLV-1 long terminal repeats (LTR) remain to be identified and characterized. RESULTS: In this study, we investigated the regulatory role of group I p21-activated kinases (Paks) in Tax-induced LTR activation. Both wild-type and kinase-dead mutants of Pak3 were capable of potentiating the activity of Tax to activate LTR transcription. The effect of Paks on the LTR was attributed to the N-terminal regulatory domain and required the action of CREB, CREB-regulating transcriptional coactivators (CRTCs) and p300/CREB-binding protein. Paks physically associated with Tax and CRTCs. Paks were recruited to the LTR in the presence of Tax. siRNAs against either Pak1 or Pak3 prevented the interaction of Tax with CRTC1 and the recruitment of Tax to the LTR. These siRNAs also inhibited LTR-dependent transcription in HTLV-1-transformed MT4 cells and in cells transfected with an infectious clone of HTLV-1. CONCLUSION: Group I Paks augment Tax-mediated transcriptional activation of HTLV-1 LTR in a kinase-independent manner.

LKB1 tumor suppressor and salt-inducible kinases negatively regulate human T-cell leukemia virus type 1 transcription.

BACKGROUND: Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia (ATL). Treatment options are limited and prophylactic agents are not available. We have previously demonstrated an essential role for CREB-regulating transcriptional coactivators (CRTCs) in HTLV-1 transcription. RESULTS: In this study we report on the negative regulatory role of LKB1 tumor suppressor and salt-inducible kinases (SIKs) in the activation of HTLV-1 long terminal repeats (LTR) by the oncoprotein Tax. Activation of LKB1 and SIKs effectively blunted Tax activity in a phosphorylation-dependent manner, whereas compromising these kinases, but not AMP-dependent protein kinases, augmented Tax function. Activated LKB1 and SIKs associated with Tax and suppressed Tax-induced LTR activation by counteracting CRTCs and CREB. Enforced expression of LKB1 or SIK1 in cells transfected with HTLV-1 molecular clone pX1MT repressed proviral transcription. On the contrary, depletion of LKB1 in pX1MT-transfected cells and in HTLV-1-transformed T cells boosted the expression of Tax. Treatment of HTLV-1 transformed cells with metformin led to LKB1/SIK1 activation, reduction in Tax expression, and inhibition of cell proliferation. CONCLUSIONS: Our findings revealed a new function of LKB1 and SIKs as negative regulators of HTLV-1 transcription. Pharmaceutical activation of LKB1 and SIKs might be considered as a new strategy in anti-HTLV-1 and anti-ATL therapy.

The centrosomal protein Tax1 binding protein 2 is a novel tumor suppressor in hepatocellular carcinoma regulated by cyclin-dependent kinase 2.

UNLABELLED: Deregulation of cellular-signaling pathways by the inactivation of tumor-suppressor genes is one of the major causes of hepatocellular carcinoma (HCC). In this study, we identified Tax1 binding protein 2 (TAX1BP2) as a novel tumor-suppressor gene in HCC. TAX1BP2 transcript was frequently underexpressed (42.2% with T/NT <0.5; P < 0.03) in HCCs, and underexpression of TAX1BP2 was associated with poorer overall survival rates in patients after surgical resection. An effector domain (ED) for TAX1BP2 tumor-suppressor activity was mapped to the amino-acid residues 267-756. Transient or stable expression of either full-length or ED of TAX1BP2 significantly suppressed HCC cell tumorigenicity through the activation of the p38/p53/p21 pathway. In contrast, silencing of TAX1BP2 by short interfering RNA remarkably suppressed the activation of the p38/p53/p21 pathway. Finally, phosphorylation of TAX1BP2 at serine-763 by cyclin-dependent kinase (CDK)2 abolished the TAX1BP2-mediated p38 activation and tumor-suppressive activity, indicating that TAX1BP2 can adapt CDK2 signaling to the p38/p53/p21 pathway. CONCLUSION: Taken together, our data provide the first evidence that TAX1BP2 is a CDK2-regulated tumor-suppressor gene in HCC and is a novel activator of the p38/p53/p21 pathway.

The retroviral oncoprotein Tax targets the coiled-coil centrosomal protein TAX1BP2 to induce centrosome overduplication.

Emerging evidence suggests that supernumerary centrosomes drive genome instability and oncogenesis. Human T-cell leukaemia virus type I (HTLV-I) is etiologically associated with adult T-cell leukaemia (ATL). ATL cells are aneuploid, but the causes of aneuploidy are incompletely understood. Here, we show that centrosome amplification is frequent in HTLV-I-transformed cells and that this phenotype is caused by the viral Tax oncoprotein. We also show that the fraction of Tax protein that localizes to centrosomes interacts with TAX1BP2, a novel centrosomal protein composed almost entirely of coiled-coil domains. Overexpression of TAX1BP2 inhibited centrosome duplication, whereas depletion of TAX1BP2 by RNAi resulted in centrosome hyperamplification. Our findings suggest that the HTLV-I Tax oncoprotein targets TAX1BP2 causing genomic instability and aneuploidy.

Garlic-derived S-allylmercaptocysteine is a hepato-protective agent in non-alcoholic fatty liver disease in vivo animal model.

PURPOSE: To investigate the hepato-protective properties and underlying mechanisms of SAMC in a non-alcoholic fatty liver disease (NAFLD) rat model. METHODS: Female rats were fed with a diet comprising highly unsaturated fat diet (30% fish oil) for 8 weeks to develop NAFLD with or without an intraperitoneal injection of 200 mg/kg SAMC three times per week. After euthanasia, blood and liver samples of rats were collected for histological and biochemical analyses. RESULTS: Co-treatment of SAMC attenuated NAFLD-induced liver injury, fat accumulation, collagen formation and free fatty acids (FFAs). At the molecular level, SAMC decreased the lipogenesis marker and restored the lipolysis marker. SAMC also reduced the expression levels of pro-fibrogenic factors and diminished liver oxidative stress partly through the inhibition in the activity of cytochrome P450 2E1-dependent pathway. NAFLD-induced inflammation was also partially mitigated by SAMC treatment via reduction in the pro-inflammatory mediators, chemokines and suppressor of cytokine signaling. The protective effect of SAMC is also shown partly through the restoration of altered phosphorylation status of FFAs-dependent MAP kinase pathways and diminished in the nuclear transcription factors (NF-κB and AP-1) activity during NAFLD development. CONCLUSIONS: SAMC is a novel hepato-protective agent against NAFLD caused by abnormal liver functions. Garlic or garlic derivatives could be considered as a potent food supplement in the prevention of fatty liver disease.

S-allylmercaptocysteine reduces carbon tetrachloride-induced hepatic oxidative stress and necroinflammation via nuclear factor kappa B-dependent pathways in mice.

PURPOSE: To study the protective effects and underlying molecular mechanisms of SAMC on carbon tetrachloride (CCl4)-induced acute hepatotoxicity in the mouse model. METHODS: Mice were intraperitoneally injected with CCl4 (50 µl/kg; single dose) to induce acute hepatotoxicity with or without a 2-h pre-treatment of SAMC intraperitoneal injection (200 mg/kg; single dose). After 8 h, the blood serum and liver samples of mice were collected and subjected to measurements of histological and molecular parameters of hepatotoxicity. RESULTS: SAMC reduced CCl4-triggered cellular necrosis and inflammation in the liver under histological analysis. Since co-treatment of SAMC and CCl4 enhanced the expressions of antioxidant enzymes, reduced the nitric oxide (NO)-dependent oxidative stress, and inhibited lipid peroxidation induced by CCl4. SAMC played an essential antioxidative role during CCl4-induced hepatotoxicity. Administration of SAMC also ameliorated hepatic inflammation induced by CCl4 via inhibiting the activity of NF-κB subunits p50 and p65, thus reducing the expressions of pro-inflammatory cytokines, mediators, and chemokines, as well as promoting pro-regenerative factors at both transcriptional and translational levels. CONCLUSIONS: Our results indicate that SAMC mitigates cellular damage, oxidative stress, and inflammation in CCl4-induced acute hepatotoxicity mouse model through regulation of NF-κB. Garlic or garlic derivatives may therefore be a potential food supplement in the prevention of liver damage.

TEC kinase stabilizes PLK4 to promote liver cancer metastasis.

Aberrated PLK4 expression has been reported in different malignancies and causes centrosome amplification, aneuploidy, and genomic instability. However, the mechanism by which PLK4 is regulated in carcinogenesis remains not fully characterised. Here, we showed that PLK4 was overexpressed in human HCC and overexpression of PLK4 predicted poorer patient prognosis. Unexpectedly, we found that induced expression of PLK4 promotes, but knockdown of PLK4 inhibits, HCC cell migration and invasion. Mechanistically, we found that TEC tyrosine kinase, which also promotes HCC cell migration, stabilizes PLK4 by phosphorylation. TEC directly phosphorylates PLK4 at tyrosine 86 residue, which not only stabilizes the protein but also enhances PLK4-mediated HCC cell invasion. Further investigation by transcriptome sequencing indicated that PLK4 promotes the phosphorylation of focal adhesion kinase to regulate the focal adhesion pathway in HCC cell migration. Taken together, our results demonstrated that PLK4 plays an important role in HCC metastasis and revealed for the first time the mechanism by which PLK4 promotes HCC metastasis via TEC phosphorylation.

Gamma-tocotrienol as an effective agent in targeting prostate cancer stem cell-like population.

Emerging evidence supports that prostate cancer originates from a rare subpopulation of cells, namely prostate cancer stem cells (CSCs). Conventional therapies for prostate cancer are believed to mainly target the majority of differentiated tumor cells but spare CSCs, which may account for the subsequent disease relapse after treatment. Therefore, successful elimination of CSCs may be an effective strategy to achieve complete remission from this disease. Gamma-tocotrienols (γ-T3) is one of the vitamin-E constituents, which have been shown to have anticancer effects against a wide range of human cancers. Recently, we have reported that γ-T3 treatment not only inhibits prostate cancer cell invasion but also sensitizes the cells to docetaxel-induced apoptosis, suggesting that γ-T3 may be an effective therapeutic agent against advanced stage prostate cancer. Here, we demonstrate for the first time that γ-T3 can downregulate the expression of prostate CSC markers (CD133/CD44) in androgen-independent prostate cancer cell lines (PC-3 and DU145), as evident from Western blotting analysis. Meanwhile, the spheroid formation ability of the prostate cancer cells was significantly hampered by γ-T3 treatment. In addition, pretreatment of PC-3 cells with γ-T3 was found to suppress tumor initiation ability of the cells. More importantly, although CD133-enriched PC-3 cells were highly resistant to docetaxel treatment, these cells were as sensitive to γ-T3 treatment as the CD133-depleted population. Our data suggest that γ-T3 may be an effective agent in targeting prostate CSCs, which may account for its anticancer and chemosensitizing effects reported in previous studies.

Effect of corticosterone and paroxetine on masculine mating behavior: possible involvement of neurogenesis.

INTRODUCTION: Corticosterone inhibits male rodent sexual behavior while the mechanism remains obscured. Recent studies have disclosed that neurogenesis in the subventricular zone (SVZ) can be increased by pheromone exposure from the opposite sex, and neurogenesis is essential for normal mating behavior of female mice. Together with the neurogenesis-inhibiting effect of corticosterone, we hypothesize that cell proliferation in the olfactory system is essential for male rodent sexual functioning. AIM: The current study explored the relationship between cell proliferation in the olfactory system and male sexual behavior. MAIN OUTCOME MEASURES: Sexual behavior performance, proliferative cell counts, and c-fos-expressing cell counts. METHODS: Adult male rats were treated with corticosterone and/or paroxetine, an antidepressant, for 2 weeks. These two drugs were shown to suppress and enhance hippocampus and SVZ cell proliferation, respectively. Mating behavior was assessed after the treatment, and proliferation of new cells and c-fos-expressing cells, activated neurons in the mating-related regions in the brain, were analyzed. To further confirm the necessity of cell proliferation in mating, inhibition of cell proliferation was performed by intracerebroventricular infusion of cytostatic cytosine arabinose (Ara-c). RESULTS: Corticosterone treatment, which inhibited cell proliferation in both the SVZ and olfactory epithelium, led to inhibited male sexual performance. In contrast, paroxetine increased cell proliferation and improved the performance in corticosterone-treated animals. When cell proliferation in the brain was inhibited by Ara-c, a suppressed sexual performance was found. However, cell proliferation in olfactory epithelium was not inhibited by Ara-c and thus the sexual inhibition is unlikely to be linked to this region. Furthermore, a decrease in c-fos expression in the mating-related regions upon female pheromone stimulation was found. CONCLUSIONS: These results suggest that cell proliferation in the SVZ and hippocampus may be involved in the reproduction of the male rodents, and pharmacological treatments may affect sexual functioning through alteration of neurogenesis.

Deleted in liver cancer 2 suppresses cell growth via the regulation of the Raf-1-ERK1/2-p70S6K signalling pathway.

BACKGROUND: Deleted in liver cancer 2 (DLC2) gene, a putative tumour suppressor gene, encodes a Rho GTPase-activating protein (RhoGAP) with GAP activity specific for RhoA. It exhibits tumour suppressor functions and inhibits tumour cell proliferation, migration as well as transformation. AIMS: In this study, we aimed to investigate the underlying mechanisms of the DLC2 gene in suppressing cell migration and cell growth. HepG2 hepatoma cells were stably transfected with the DLC2γ isoform, which contains the RhoGAP domain. METHODS AND RESULTS: On performing immunofluorescence staining and Western blot analysis, the expression of the focal adhesion protein paxillin was found to be much reduced in DLC2γ-stable clones. Upon flow cytometric analysis of the cell cycle profiles, the DLC2γ-stable clones were shown to have a higher population of cells arrested at the G1 phase than the EGFP vector-stable clone, suggesting that downregulation of RhoA activity in DLC2γ-stable clones inhibited cell cycle progression. In the DLC2γ-stable clone, the levels of Raf-1 and extracellular signal-regulated kinase (ERK) 1/2 were decreased as compared with those of the parental HepG2, EGFP vector and DLC2γ-GAP defective mutant-stable clones. Furthermore, the ribosomal kinase p70S6K, a downstream target of ERK1/2, was suppressed in the DLC2-stable clones. On the contrary, when DLC2 was knocked down by siRNA in HepG2 cells, the expression levels of phospho-p70S6K and phospho-ERK1/2 were upregulated. CONCLUSION: Our data show that DLC2 inhibits the activity of Raf-1-ERK1/2-p70S6K via its RhoGAP function, resulting in the suppression of cell growth. Further studies on the molecular signalling between DLC2 and p70S6K may provide an insight into its growth suppressor function.

Rapamycin and CCI-779 inhibit the mammalian target of rapamycin signalling in hepatocellular carcinoma.

BACKGROUND: The mammalian target of rapamycin (mTOR), which phosphorylates p70S6K and 4EBP1 and activates the protein translation process, is upregulated in cancers and its activation may be involved in cancer development. AIMS: In this study, we investigated the tumour-suppressive effects of rapamycin and its new analogue CCI-779 on hepatocellular carcinoma (HCC). METHODS: Rapamycin and its new analogue CCI-779 were applied to treat HCC cells. Cell proliferation, cell cycle profile and tumorigenicity were analysed. RESULTS: In human HCCs, we observed frequent (67%, 37/55) overexpression of mTOR transcripts using real-time reverse transcriptase-polymerase chain reaction. Upon drug treatment, PLC/PRF/5 showed the greatest reduction in cell proliferation using the colony formation assay, as compared with HepG2, Hep3B and HLE. Rapamycin was a more potent antiproliferative agent than CCI-779 in HCC cell lines. Proliferation assays by cell counting showed that the IC(50) value of rapamycin was lower than that of CCI-779 in PLC/PRF/5 cells. Furthermore, flow cytometric analysis showed that both drugs could arrest HCC cells in the G(1) phase but did not induce apoptosis of these cells, suggesting that these mTOR inhibitors are cytostatic rather than cytotoxic. Upon rapamycin and CCI-779 treatment, the phosphorylation level of mTOR and p70S6K in HCC cell lines was significantly reduced, indicating that both drugs can suppress mTOR activity in HCC cells. In addition, both drugs significantly inhibited the growth of xenografts of PLC/PRF/5 cells in nude mice. CONCLUSIONS: Our findings indicate that rapamycin and its clinical analogue CCI-779 possess tumour-suppressive functions towards HCC cells.

Centrosomal protein TAX1BP2 inhibits centrosome-microtubules aberrations induced by hepatitis B virus X oncoprotein.

Liver cancer (hepatocellular carcinoma, HCC) is one of the most prevalent cancers worldwide. Several etiological factors of HCC, including hepatitis B or hepatitis C virus infection, liver cirrhosis and aflatoxin B1 intake has been identified. HBx, which is an oncogenic protein encoded by the hepatitis B virus, is strongly associated with hepatocarcinogenesis. Using stable HBx-expressing cell, we showed that HBx induced chromosome gain, with amplification of centrosomes numbers and deregulation of centrosome ultrastructure. To dissect the mechanism for chromosome instability, our result revealed that HBx contributed to a hyperactive centrosome-microtubule dynamics by accelerating microtubule nucleation and polymerization. Further investigations suggested that HBx interacted with a centrosome linker protein TAX1BP2, which has previously been shown to function as an intrinsic block of centrosome amplification and a tumour suppressor in HCC. Restoring TAX1BP2 was able to block HBx-mediated centrosome amplification and abolish the HBx-mediated centrosome aberration, thereby suppressing chromosome instability. Thus, we demonstrate here a mechanism by which HBx deregulates centrosome-microtubule dynamics through interacting with TAX1BP2, which underlines the possibility of restoration of TAX1BP2 to rescue cells from chromosome instability.

HAI-2 is epigenetically downregulated in human hepatocellular carcinoma, and its Kunitz domain type 1 is critical for anti-invasive functions.

Pharmacological demethylation-based gene expression profile analysis is a useful tool to identify epigenetically silenced tumour suppressor genes. HGF activator inhibitor 2 (HAI-2), a serine protease inhibitor, has been identified as one of the candidate tumour suppressor genes in human hepatocellular carcinoma (HCC) with this technique. In this study, we aimed to characterise the epigenetic status and tumour suppressive function of HAI-2 in HCC. We validated that HAI-2 expression was either absent or low in most of the HCC cell lines tested, and 5-Aza-2'-deoxycytidine treatment significantly restored its expression in 9 (75%) of these 12 cell lines. HAI-2 was found to be frequently underexpressed in human HCCs (p < 0.001). With bisulphite DNA sequencing and methylation-specific PCR, we found that the promoter of the HAI-2 gene was frequently hypermethylated in both HCC cell lines and human HCCs. Ectopic expression of HAI-2 significantly inhibited cell migration and invasiveness of HCC cells in vitro and suppressed tumourigenicity in vivo. In addition, we also provided the first evidence that HAI-2 mediated its tumour suppressor function via the Kunitz domain 1 (KD-1), as KD-1 but not KD-2 inactivating mutant abolished its anti-tumour invasiveness in vitro. Our findings suggest that HAI-2 is a candidate tumour suppressor gene that is frequently hypermethylated and underexpressed in human HCCs, and the KD-1 domain of HAI-2 is the key region responsible for its anti-invasive function.

Berberine inhibits Rho GTPases and cell migration at low doses but induces G2 arrest and apoptosis at high doses in human cancer cells.

Berberine is an active ingredient extracted from Coptidis rhizoma which has been used for centuries as a traditional Chinese medicine for treatment of inflammatory diseases. Recent studies have indicated that berberine has anticancer properties. Berberine arrested cell growth and inhibited cell migration in various cancer cell lines. In this study, we examined the effects of berberine on HONE1 cells, which have been commonly used as a cell model for nasopharyngeal carcinoma. We observed the inhibitory effects of berberine on HONE1 cells at a high dosage (>150 microM). Berberine effectively induced the mitotic arrest of HONE1 cells at 300 microM which was associated with apoptosis. Berberine had differential intracellular localization at low and high doses. At a low dose (50 microM), berberine was localized in the mitochondria while at a high dose (300 microM), berberine was localized in the nucleus which may have induced mitotic arrest. Berberine effectively inhibited cell migration and invasion at low doses. Using a specific GST pull-down assay of activated Rho GTPases, we demonstrated that berberine suppressed the activation of Rho GTPases including RhoA, Cdc42 and Rac1. This indicates a novel function of berberine in the suppression of Rho GTPase signaling to mediate its inhibitory action on cell migration and motility. The potential of berberine to inhibit cancer metastasis in cancer warrants further investigation.

P21-activated protein kinase is overexpressed in hepatocellular carcinoma and enhances cancer metastasis involving c-Jun NH2-terminal kinase activation and paxillin phosphorylation.

Hepatocellular carcinoma (HCC) is one of the major malignancies in the world. The prognosis of HCC is poor, due to frequent intrahepatic metastasis and tumor recurrence. P21-activated protein kinase (Pak1), a main downstream effector of small Rho GTPases, Rac1 and Cdc42, plays an important role in the regulation of cell morphogenesis, motility, mitosis, and angiogenesis. Here, we show that Pak1 gene was overexpressed in human HCCs. Overexpression of Pak1 in human HCCs was associated with more aggressive tumor behavior in terms of more metastatic phenotype and more advanced tumor stages. In addition, HCC cell line stably expressing Pak1 displayed increased cell motility rates and, conversely, knockdown of endogenous Pak1 expression by small interfering RNA reduced the migration rates of HCC cells. In an established metastatic HCC cell line, we found that Pak1 was overexpressed compared with its primary HCC cell line and this overexpression was associated with higher cell motility. Importantly, we found that c-Jun NH(2)-terminal kinase (JNK) was activated in HCC cell lines overexpressing Pak1. Inhibition of the JNK activity by chemical inhibitor significantly reduced the migration rates of HCC cells via attenuation of paxillin phosphorylation at Ser(178). In conclusion, our results document that Pak1 is overexpressed in HCCs and plays an important role in the metastasis of HCC. The mechanism by which Pak1 induces cancer metastasis may involve activation of JNK and phosphorylation of paxillin.