H1.0 induces paclitaxel-resistance genes expression in ovarian cancer cells by recruiting GCN5 and androgen receptor.

More than 90% of ovarian cancer deaths are due to relapse following development of chemoresistance. Our main objective is to better understand the molecular mechanism underlying paclitaxel resistance (taxol resistance, Txr) in ovarian cancer. Here, we observed that the linker histone H1.0 is upregulated in paclitaxel-resistant ovarian cancer cells. Knockdown of H1.0 significantly downregulates the androgen receptor (AR) and sensitizes paclitaxel-resistant SKOV3/Txr and 2774/Txr cell lines to paclitaxel. Conversely, ectopic expression of H1.0 upregulates AR and increases Txr in parental SKOV3 and MDAH2774 cells. Notably, H1.0 upregulation is associated with disease recurrence and poor survival in a subset of ovarian cancer subjects. Inhibition of PI3K significantly reduces H1.0 mRNA and protein levels in paclitaxel-resistant cells, suggesting the involvement of the PI3K/AKT signaling pathway. Knockdown of H1.0 and AR also downregulates the Txr genes ABCB1 and ABCG2 in paclitaxel-resistant cells. Our data show that H1.0 induces GCN5 expression and histone acetylation, thereby enhancing Txr gene transactivation. These findings suggest that Txr in ovarian cancer involves the PI3K/AKT pathway and leads to upregulation of histone H1.0, recruitment of GCN5 and AR, followed by upregulation of a subgroup of Txr genes that include ABCB1 and ABCG2. This study is the first report describing the relationship between histone H1.0 and GCN5 that cooperate to induce AR-dependent Txr in ovarian cancer cells.

Androgen receptor transcriptional activity and chromatin modifications on the ABCB1/MDR gene are critical for taxol resistance in ovarian cancer cells.

We report here that the androgen receptor (AR) and ABCB1 are upregulated in a model of acquired taxol resistance (txr) in ovarian carcinoma cells. AR silencing sensitizes txr cells to taxol threefold, whereas ectopic AR expression in AR-null HEK293 cells induces resistance to taxol by 1.7-fold. AR activation using the agonist dihydrotestosterone (DHT) or sublethal taxol treatment upregulates ABCB1 expression in both txr cells and AR-expressing HEK293 cells. In contrast, AR inactivation using the antagonist bicalutamide downregulates ABCB1 expression and enhances cytotoxicity to taxol. A functional ABCB1 promoter containing five predicted androgen-response elements (AREs) is cloned. Deletion assays reveal a taxol-responsive promoter segment which harbors ARE4. Notably, DHT- or taxol-activated AR potentiates binding of the AR to ARE4 as revealed by the chromatin immunoprecipitation. On the other hand, txr cells display an increase in chromatin remodeling. AR/H3K9ac and AR/H3K14ac complexes bind specifically to ARE4 in response to taxol. Furthermore, acetyltransferase protein levels (p300 and GCN5) are upregulated in txr cells. Silencing of p300 or GCN5 reduces chromatin modification and enhances cytotoxicity in both parental and txr SKOV3 cells. While the phosphatidylinositol 3-kinase (PI3K)/serine/threonine protein kinase (AKT) pathway is significantly activated by taxol, taxol-induced ABCB1 expression, histone posttranslational modifications, and p300 binding to ARE4 are suppressed following inhibition of the PI3K/AKT cellular pathway. These results demonstrate that the AKT/p300/AR axis can be activated to target ABCB1 gene expression in response to taxol, thus revealing a new treatment target to counter taxol resistance.

TLR4 and NFκB signaling is critical for taxol resistance in ovarian carcinoma cells.

We report here that toll-like receptor 4 (TLR4) and ABCB1 are upregulated in SKOV3 ovarian carcinoma cells that acquired resistance to the anticancer drug taxol. Silencing of TLR4 using short-hairpin RNA sensitized taxol-resistant SKOV3 cells to taxol (4.6 fold), whereas ectopic expression of TLR4 in parental, taxol-sensitive SKOV3 cells or TLR4-null HEK293 cells induced taxol resistance (∼2 fold). A sub-lethal dose of taxol induced ABCB1 protein expression in taxol-resistant SKOV3 cells. Inactivation of TLR4 using chemical inhibitors (CLI-095 and AO-I) downregulated ABCB1 protein expression and enhanced the cytotoxic activity of taxol in taxol-resistant SKOV3 cells. While the sensitization effect of TLR4 inactivation was also detected in TOV21G ovarian cancer cells, which express moderate level of TLR4, ectopic expression of ABCB1 prevented the sensitization effect in these cells. Notably, the NFκB pathway was significantly activated by taxol, and inhibition of this pathway suppressed TLR4-regulated ABCB1 expression. Furthermore, taxol-induced NFκB signaling was reduced following TLR4 silencing in taxol-resistant SKOV3 cells. Consistent with these results, ectopic expression of TLR4 in taxol-sensitive SKOV3 cells enhanced ABCB1 expression and conferred resistance to taxol. The protective effect of exogenous TLR4 expression against taxol was reduced by treatment with NFκB inhibitor in these cells. These results demonstrate that taxol activates the TLR4-NFκB pathway which in turn induces ABCB1 gene expression. This cellular pathway thus represents a novel target to limit resistance to taxol in ovarian cancer cells.

Targeting HSP60 by subcutaneous injections of jetPEI/HSP60-shRNA destabilizes cytoplasmic survivin and inhibits hepatocellular carcinoma growth.

Heat shock protein 60 (HSP60) overexpresses in various types of cancer, but its expression levels and functions in hepatocellular carcinoma (HCC) are still in dispute. We aim to clarify this issue and examine whether HSP60 could be a therapeutic target for HCC. We found drastically enhanced cell apoptosis and suppressed cell proliferation in two HCC cell lines with HSP60-silencing, and also indicated survivin was involved in this regulatory process in vitro and in vivo. However, HSP60-silencing in normal human hepatocytes only resulted in a minimal reduction of cell proliferation but without effects on cell apoptosis. We also showed HSP60 interacted with cytosolic but not mitochondrial survivin by immunoprecipitation assay. A rigorous method was used to standardize quantification from immunoblot assay to obtain more precise expression levels of HSP60 and survivin. The expression of HSP60 and survivin positively correlated in both cancerous and non-cancerous liver tissues (P < 0.001) after analyzing 145 surgically removed HCC tissues. A total of 56.6% of HCC patients overexpressed HSP60 in cancerous tissues, and 40.0% under-expressed HSP60. Higher expression of HSP60 and survivin in non-cancerous tissues both correlated with shorter overall survival (P = 0.029 and P < 0.001, respectively). Finally, we evaluated the therapeutic potential of HSP60 using extraneous delivery of jetPEI/shHSP60 complexes. The treatment results showed significant reduction of tumor weight by 44.3% (P < 0.05), accompanied by under-expression of survivin. These studies suggested that HSP60 not only served as a prognostic marker but also served as a novel therapeutic target for HCC.

CITED2 silencing sensitizes cancer cells to cisplatin by inhibiting p53 trans-activation and chromatin relaxation on the ERCC1 DNA repair gene.

In this study, we show that silencing of CITED2 using small-hairpin RNA (shCITED2) induced DNA damage and reduction of ERCC1 gene expression in HEK293, HeLa and H1299 cells, even in the absence of cisplatin. In contrast, ectopic expression of ERCC1 significantly reduced intrinsic and induced DNA damage levels, and rescued the effects of CITED2 silencing on cell viability. The effects of CITED2 silencing on DNA repair and cell death were associated with p53 activity. Furthermore, CITED2 silencing caused severe elimination of the p300 protein and markers of relaxed chromatin (acetylated H3 and H4, i.e. H3K9Ac and H3K14Ac) in HEK293 cells. Chromatin immunoprecipitation assays further revealed that DNA damage induced binding of p53 along with H3K9Ac or H3K14Ac at the ERCC1 promoter, an effect which was almost entirely abrogated by silencing of CITED2 or p300. Moreover, lentivirus-based CITED2 silencing sensitized HeLa cell line-derived tumor xenografts to cisplatin in immune-deficient mice. These results demonstrate that CITED2/p300 can be recruited by p53 at the promoter of the repair gene ERCC1 in response to cisplatin-induced DNA damage. The CITED2/p300/p53/ERCC1 pathway is thus involved in the cell response to cisplatin and represents a potential target for cancer therapy.

Repurposing of phentolamine as a potential anticancer agent against human castration-resistant prostate cancer: A central role on microtubule stabilization and mitochondrial apoptosis pathway.

BACKGROUND: Drug repurposing of phentolamine, an α-adrenoceptor antagonist, as an anticancer agent has been studied in human castration-resistant prostate cancer (CRPC). METHODS: Cell proliferation was examined by sulforhodamine B and CFSE staining assays. Cell cycle progression and mitochondrial membrane potential (ΔΨm) were detected by flow cytometric analysis. Protein expression was detected by Western blotting. Effect on tubulin/microtubule was determined using confocal immunofluorescence microscopic examination, microtubule assembly detection, tubulin turbidity assay, and binding assay. Several assessments were used to characterize apoptotic signaling pathways and combinatory effect. RESULTS: Phentolamine induced anti-proliferative effect in PC-3 and DU-145, two CRPC cell lines, and P-glycoprotein (P-gp) overexpressing cells. This effect was not significantly reduced in paclitaxel-resistant cells. Rhodamine 123 efflux assay showed that phentolamine was not a P-gp substrate. Phentolamine induced mitotic arrest of the cell cycle and formation of hyperdiploid cells, followed by an increase of apoptosis. Mitotic arrest was confirmed by cyclin B1 up-regulation, Cdk1 activation, and a dramatic increase of mitotic protein phosphorylation. Both in vitro and cellular identification demonstrated that phentolamine, similar to paclitaxel, induced tubulin polymerization and formation of multiple nuclei. Besides, it did not compete with paclitaxel binding on tubulin. Phentolamine induced the phosphorylation and degradation of Bcl-2 and Bcl-xL, two anti-apoptotic Bcl-2 family members, and the loss of ΔΨm indicating the induction of mitochondrial damage. It ultimately induced the activation of caspase-9, -8, and -3 and apoptotic cell death. Moreover, combination treatment with phentolamine and paclitaxel caused a synergistic apoptosis. CONCLUSIONS: The data suggest that phentolamine is a potential anticancer agent. In contrast to a wide variety of microtubule disrupting agents, phentolamine induces microtubule assembly, leading to mitotic arrest of the cell cycle which "in turn" induces subsequent mitochondrial damage and activation of related apoptotic signaling pathways in CRPC cells. Furthermore, combination between phentolamine and paclitaxel induces a synergistic apoptotic cell death. Phentolamine has a simple chemical structure and is not a P-gp substrate. Optimization of phentolamine structure may also be a potential approach for further development.

EBV-encoded LMP-1 sensitizes nasopharyngeal carcinoma cells to genotoxic drugs by down-regulating Cabin1 expression.

The oncogenic latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is involved in the pathogenesis of human nasopharyngeal carcinoma (NPC) and lymphoma. We and other authors have shown earlier that LMP1 induces apoptosis and inhibits xenograft tumor growth in mice, but the mechanism underlying these processes has not been investigated so far. In the present study, we show that knockdown of LMP1 renders the EBV-positive NPC cell line CG-1 resistant to various genotoxic drugs (cisplatin, etoposide, and adriamycin). LMP1 inhibits the expression of Cabin1, a Ca(2+) regulated protein shown earlier to inhibit calcineurin. Knockdown of calcineurin binding protein (Cabin1) with small hairpin RNA sensitizes CG-1 cells to genotoxic drugs. In contrast, LMP1 overexpression reduces Cabin1 level and renders both CG-1 cells and EBV-negative NPC cell lines sensitive to cisplatin. The c-Jun-N-terminal kinase (JNK) and ERK pathways are required for LMP1-induced suppression of Cabin1 at the transcriptional level. Chromatin immunoprecipitation assays further confirm that the JNK-activated transcription factor AP-1 mediates the LMP1-induced down-regulation of Cabin1 gene expression. LMP1 knockdown also increases the resistance of xenograph tumors to cisplatin in mice, therefore confirming the relevance of our findings in vivo. This study reveals the molecular mechanism underlying the pro-apoptotic activity of LMP1 during cisplatin-based NPC chemotherapy.

Sorafenib induces endometrial carcinoma apoptosis by inhibiting Elk-1-dependent Mcl-1 transcription and inducing Akt/GSK3ß-dependent protein degradation.

Endometrial carcinoma (EC) is one of the main gynecologic malignancies affecting women, but effective treatments are currently lacking. In the present study, we investigated the effect of sorafenib, a general kinase inhibitor, on several EC cell lines (HEC1A, HEC1B, and RL95-2). Sorafenib induced cell death in EC cells with the following order of sensitivity: HEC1A > HEC1B > RL95-2. Sorafenib suppressed several anti-apoptotic proteins in HEC1A cells, including myeloid cell leukemia 1 (Mcl-1). Ectopic overexpression of Mcl-1 prevented the cell killing effect of sorafenib. Sorafenib suppressed Mcl-1 at the gene transactivation level by inactivating the ERK/Elk-1 pathway. Accordingly, the inhibitory effect of sorafenib on Mcl-1 expression decreased following knockdown of Elk-1 using short-hairpin RNA (shRNA). Elk-1 overexpression rescued both the inhibitory effect of sorafenib on Mcl-1 expression and the cell killing effect of sorafenib. Furthermore, sorafenib reduced the stability of the Mcl-1 protein by enhancing its ubiquitination and degradation by the proteasome via the AKT/GSK3ß and the ERK pathways. Similar results were detected in other EC cell lines. These results indicate that sorafenib induces apoptosis in EC cells by down-regulating the anti-apoptotic protein Mcl-1 via transcriptional inhibition and protein degradation. Our results thus support the notion that sorafenib may be used in endometrial cancer therapy.

Identification and functional characterization of zebrafish Gas7 gene in early development.

Growth arrest-specific 7 (Gas7) is preferentially expressed in the nervous system and plays an important role during neuritogenesis in mammals. However, the structure and function of Gas7 homologs have not been studied in nonmammalian vertebrates used as models. In this report, we identify a Gas7 gene in zebrafish that we termed zfGas7. The transcript of this gene was produced by canonical splicing, and its protein product contained a Fes/CIP4 homology and a coiled-coil domain. In early zebrafish embryos, RT-PCR analyses revealed that zfGas7 was initially expressed at 5.3 hr postfertilization (hpf), followed by an increase of expression at 10 hpf and further accumulation during somitogenesis at 48 hpf. Spatiotemporal analyses further showed that Gas7 mRNA was detected in the brain, somite, and posterior presomitic mesoderm regions during somitogenesis. At 36 hpf, zfGas7 mRNA was detected in the brain and somite but was later found only in neuronal clusters of the brain at 52 hpf. Gas7 knockdown with morpholino antisense oligonucleotides (Gas7MO) reduced the number of HuC-positive neurons in the trigeminal and statoacoustic ganglions and produced deformed phenotypes, such as flattening of the top of the head. Notably, the neuron reduction and deformed phenotypes observed in Gas7MO embryos were partially rescued by ectopic expression of Gas7. Because altered somitogenesis and pigmentation were also found in the morphants, the neuronal phenotypes observed likely are due to a general developmental delay of embryogenesis. These results indicate that Gas7 is expressed in neuronal cells but is not specifically required for neuronal development in vertebrates.

Golgi-SNARE GS28 potentiates cisplatin-induced apoptosis by forming GS28-MDM2-p53 complexes and by preventing the ubiquitination and degradation of p53.

In the present study, we observed that the Golgi-SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) GS28 forms a complex with p53 in HEK (human embryonic kidney)-293 cells. Given that p53 represents a tumour suppressor that affects the sensitivity of cancer cells to various chemotherapeutic drugs, we examined whether GS28 may influence the level of sensitivity to the DNA-damaging drug cisplatin. Indeed, knockdown of GS28 using short-hairpin RNA (shGS28) induced resistance to cisplatin in HEK-293 cells. On the other hand, overexpression of GS28 sensitized HEK-293 cells to cisplatin, whereas no sensitization effect was noted for the mitotic spindle-damaging drugs vincristine and taxol. Accordingly, we observed that knockdown of GS28 reduced the accumulation of p53 and its pro-apoptotic target Bax. Conversely, GS28 overexpression induced the accumulation of p53 and Bax as well as the pro-apoptotic phosphorylation of p53 on Ser(46). Further experiments showed that these cellular responses could be abrogated by the p53 inhibitor PFT-α (pifithrin-α), indicating that GS28 may affect the stability and activity of p53. The modulatory effects of GS28 on cisplatin sensitivity and p53 stability were absent in lung cancer H1299 cells which are p53-null. As expected, ectopic expression of p53 in H1299 cells restored the modulatory effects of GS28 on sensitivity to cisplatin. In addition, GS28 was found to form a complex with the p53 E3 ligase MDM2 (murine double minute 2) in H1299 cells. Furthermore, the ubiquitination of p53 was reduced by overexpression of GS28 in cells, confirming that GS28 enhances the stability of the p53 protein. Taken together, these results suggest that GS28 may potentiate cells to DNA-damage-induced apoptosis by inhibiting the ubiquitination and degradation of p53.

Knockdown of CITED2 using short-hairpin RNA sensitizes cancer cells to cisplatin through stabilization of p53 and enhancement of p53-dependent apoptosis.

CITED2 is a transcriptional modulator which has been implicated in human oncogenesis. In the present study, we examined whether CITED2 is also involved in the resistance of cancer cells to the chemotherapeutic drug cisplatin. We first observed that knockdown of CITED2 using short-hairpin RNA sensitized non-tumorigenic HEK293 cells to cisplatin. Sensitization to cisplatin following knockdown of CITED2 was also observed in cervical carcinoma HeLa cells and in cisplatin-resistant HeLa cells, thereby showing that acquired cisplatin resistance could be reversed by CITED2 knockdown. This sensitization response was dependent on the status of p53 since efficient sensitization was observed in p53-positive hepatocellular carcinoma (HCC) Sk-Hep-1 cells, whereas a negligible response was produced in the two p53-defective cell lines HCC Mahlavu and lung cancer H1299. In contrast, overexpression of CITED2 decreased sensitivity of HEK293 cells to cisplatin, while moderate resistance was produced in HeLa cells. Overexpression of CITED2 also decreased sensitivity to cisplatin in p53-defective H1299 cells when exogenous p53 expression was re-introduced. We observed that knockdown of CITED2-induced CBP/p300-mediated p53 acetylation (Lys373) in HEK293 cells, thereby leading to a decrease of p53 ubiquitination and subsequent accumulation of the p53 protein. Notably, the effects of CITED2 knockdown on p53 accumulation and the increase of p53's target Bax were more pronounced after treatment with cisplatin. Based on these results, we propose that a combination of cisplatin and CITED2 shRNA may represent an effective treatment against p53-sensitive cancer cells.

TLR4/IL-6/IRF1 signaling regulates androgen receptor expression: A potential therapeutic target to overcome taxol resistance in ovarian cancer.

Ovarian cancer is poorly treatable due, at least in part, to induced drug resistance to taxol- and cisplatin-based chemotherapy. Recent studies showed that ectopic overexpression of toll-like receptor 4 (TLR4) in ovarian cancer cells leads to upregulation of the androgen receptor (AR) and transactivation of taxol resistance genes, thereby causing chemoresistance. In the present study, we examined the signaling pathways involving TLR4 and interleukin 6 (IL-6) that enhance AR expression. Based on transcriptomic analysis, we show that IL-6 functions as a hub gene among the upregulated genes in taxol-treated TLR4-overexpressing ovarian cancer cells. Both the TLR4 activator taxol and IL-6 can induce AKT phosphorylation, whereas TLR4 knockdown or inhibition of the IL-6 signal transducer GP130 abrogates AKT activation. Furthermore, expression of AR and IL-6 is downregulated in TLR4-knockdown, taxol-resistant cells. In addition, TLR4 knockdown inhibits GP130 and IL-6 receptor alpha (IL6Rα) activities, indicating that TLR4 plays a critical role in IL-6 signaling. On the other hand, nuclear translocation of AR is induced by IL-6 treatment, whereas knockdown of endogenous IL-6 reduces AR and TLR4 expression in taxol-resistant ovarian cancer cells. These results indicate that TLR4 and IL-6 play a crucial role in AR gene regulation and function. We also identify interferon regulatory factor 1 (IRF1) as a downstream target of IL-6 signaling and as a regulator of AR expression. Moreover, analysis of clinical samples indicates that high IL-6 expression correlates with poor progression-free survival in ovarian cancer patients treated with taxol. Overall, our findings indicate that the TLR4/IL-6/IRF1 signaling axis represents a potential therapeutic target to overcome AR-based taxol resistance in ovarian cancer.

Knockdown of growth-arrest-specific gene 7b (gas7b) using short-hairpin RNA desensitizes neuroblastoma cells to cisplatin: Implications for preventing apoptosis of neurons.

Efficient control of cell survival and cell proliferation is critical for the development of neuron cells. Earlier, we observed that growth arrest-specific gene 7 (Gas7) plays a role in controlling neuritogenesis in mammals. In the present study, we report that the Gas7b isoform is involved in controlling growth arrest and apoptosis of neuroblastoma cells in response to various stimuli. Accordingly, knockdown of Gas7b using small-hairpin RNA (shRNA) was shown to reduce apoptosis induced either by serum starvation or by the antineoplastic agents cisplatin and nocodazole in human neuroblastoma SH-SY5Y cells. Gas7b knockdown also enhanced the ability of the treated cells to form clones in response to cisplatin. On the other hand, forced expression of Gas7a or Gas7b isoform in mouse neuroblastoma Neuro2A cells, which express a defective Gas7 gene, rendered the cells proapoptotic and vulnerable to cisplatin-induced apoptosis. In addition, Neuro2A cells that overexpressed Gas7 showed a reduced ability to form clones. Overexpression of Gas7 produced similar but less extensive effects in nonneuronal HEK293 cells. Taken together, our observations suggest that Gas7b is involved not only in neuritogenesis but also in the regulation of neuronal cell death.

Damaged DNA-binding protein 2 (DDB2) protects against UV irradiation in human cells and Drosophila.

BACKGROUND: We observed previously that cisplatin-resistant HeLa cells were cross-resistant to UV light due to accumulation of DDB2, a protein implicated in DNA repair. More recently, we found that cFLIP, which represents an anti-apoptotic protein whose level is induced by DDB2, was implicated in preventing apoptosis induced by death-receptor signaling. In the present study, we investigated whether DDB2 has a protective role against UV irradiation and whether cFLIP is also involved in this process. METHODS: We explored the role of DDB2 in mediating UV resistance in both human cells and Drosophila. To do so, DDB2 was overexpressed by using a full-length open reading frame cDNA. Conversely, DDB2 and cFLIP were suppressed by using antisense oligonucleotides. Cell survival was measured using a colony forming assay. Apoptosis was monitored by examination of nuclear morphology, as well as by flow cytometry and Western blot analyses. A transcription reporter assay was also used to assess transcription of cFLIP. RESULTS: We first observed that the cFLIP protein was upregulated in UV-resistant HeLa cells. In addition, the cFLIP protein could be induced by stable expression of DDB2 in these cells. Notably, the anti-apoptotic effect of DDB2 against UV irradiation was largely attenuated by knockdown of cFLIP with antisense oligonucleotides in HeLa cells. Moreover, overexpression of DDB2 did not protect against UV in VA13 and XP-A cell lines which both lack cFLIP. Interestingly, ectopic expression of human DDB2 in Drosophila dramatically inhibited UV-induced fly death compared to control GFP expression. On the other hand, expression of DDB2 failed to rescue a different type of apoptosis induced by the genes Reaper or eiger. CONCLUSION: Our results show that DDB2 protects against UV stress in a cFLIP-dependent manner. In addition, the protective role of DDB2 against UV irradiation was found to be conserved in divergent living organisms such as human and Drosophila. In addition, UV irradiation may activate a cFLIP-regulated apoptotic pathway in certain cells.

Role of the TLR4-androgen receptor axis and genistein in taxol-resistant ovarian cancer cells.

Toll-like receptor 4 (TLR4) is often overexpressed in taxol-resistant cancer cells. Here we used whole-genome transcriptomic analysis to identify 787 upregulated genes in SKOV3 ovarian carcinoma cells that ectopically express TLR4. Using chromatin immunoprecipitation enrichment analysis, we observed that 27.8% of the TLR4-upregulated genes identified were androgen receptor (AR)-regulated genes. Accordingly, AR expression was induced in taxol-resistant SKOV3 cells overexpressing TLR4, whereas depletion of TLR4 by shRNA repressed AR expression. Activation of AR by androgens or silencing of AR using shRNA also regulated expression of AR-related genes. We found that expression of DCDC2, ANKRD18B, ALDH1A1, c14orf105, ITGBL1 and NEB was overexpressed in taxol-resistant cells, suggesting the involvement of these AR-related genes in taxol resistance. Pathway enrichment analysis confirmed that the expression of several upregulated genes enriched in steroid biosynthesis pathways was inducible by androgens, supporting the results of previous studies. We also observed that genistein inhibits AR activation, leading to suppression of AR-driven genes and reduced taxol resistance in ovarian cancer cells. Overall, we identified six TLR4- and AR-regulated genes involved in taxol resistance. Our results reveal that the TLR4/AR axis plays a critical role in taxol resistance and that genistein is a candidate compound to limit chemoresistance and improve cancer treatment in ovarian cancer.

Hepatitis B Virus X Protein Induces RHAMM-Dependent Motility in Hepatocellular Carcinoma Cells via PI3K-Akt-Oct-1 Signaling.

Chronic hepatitis B virus (HBV) infection is a major risk factor for the development of hepatocellular carcinoma (HCC), which represents one of the most common cancers worldwide. Recent studies suggest that HBV's protein X (HBx) plays a crucial role in HCC development and progression. Earlier, genome-wide analysis identified that the receptor for hyaluronan-mediated motility (RHAMM) represents a putative oncogene and is overexpressed in many human cancers, including HCC. However, the mechanism underlying RHAMM upregulation and its role in tumorigenesis remain unclear. Here, we show that ectopic expression of HBx activates the PI3K/Akt/Oct-1 pathway and upregulates RHAMM expression in HCC cells. HBx overexpression leads to dissociation of C/EBPß from the RHAMM gene promoter, thereby inducing RHAMM upregulation. RHAMM knockdown attenuates HBx-induced cell migration and invasion in vitro. In mice, HBx promotes cancer cell colonization via RHAMM upregulation, resulting in enhanced metastasis. Analysis of gene expression datasets reveals that RHAMM mRNA level is upregulated in patients with HCC with poor prognosis. IMPLICATIONS: These results indicate that RHAMM expression is upregulated by HBx, a process that depends on the inhibition of C/EBPß activity and activation of the PI3K/Akt/Oct-1 pathway. These results have several implications for the treatment of HBV-positive HCC involving upregulation of RHAMM and cancer metastasis. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/18/3/375/F1.large.jpg.

Inhibition of apoptosis by oncogenic hepatitis B virus X protein: Implications for the treatment of hepatocellular carcinoma.

Hepatitis B virus X protein (HBx) plays an important role in the development of hepatocellular carcinoma (HCC). In addition, hepatoma upregulated protein (HURP) is a cellular oncogene that is upregulated in a majority of HCC cases. We highlight here recent findings demonstrating a link between HBx, HURP and anti-apoptosis effects observed in cisplatin-treated HCC cells. We observed that Hep3B cells overexpressing HBx display increased HURP mRNA and protein levels, and show resistance to cisplatin-induced apoptosis. Knockdown of HURP in HBx-expressing cells reverses this effect, and sensitizes cells to cisplatin. The anti-apoptotic effect of HBx requires activation of the p38/MAPK pathway as well as expression of SATB1, survivin and HURP. Furthermore, silencing of HURP using short-hairpin RNA promotes accumulation of p53 and reduces cell proliferation in SK-Hep-1 cells (p53(+/-)), whereas these effects are not observed in p53-mutant Mahlavu cells. Similarly, HURP silencing does not affect the proliferation of H1299 lung carcinoma cells or Hep3B HCC cells which lack p53. Silencing of HURP sensitizes SK-Hep-1 cells to cisplatin. While HURP overexpression promotes p53 ubiquitination and degradation by the proteasome, HURP silencing reverses these effects. Inoculation of SK-Hep-1 cancer cells in which HURP has been silenced produces smaller tumors than control in nude mice. Besides, gankyrin, a positive regulator of the E3 ubiquitin ligase MDM2, is upregulated following HURP expression, and silencing of gankyrin reduces HURP-mediated downregulation of p53. In addition, we observed a positive correlation between HURP and gankyrin protein levels in HCC patients (r (2) = 0.778; n = 9). These findings suggest a role for the viral protein HBx and the host protein HURP in preventing p53-mediated apoptosis during cancer progression and establishment of chemoresistance.

Inhibition of JNK and prothymosin-alpha sensitizes hepatocellular carcinoma cells to cisplatin.

Cisplatin is a potent chemotherapeutic drug widely used for the treatment of human cancer. However, its efficacy against hepatocellular carcinoma (HCC) is poor for reasons that remain unclear. We show here that prothymosin-alpha (PTMA) is overexpressed in HCC cell lines. Silencing PTMA using short-hairpin RNA sensitizes HCC cells to cisplatin, while ectopic expression of PTMA induces cell resistance to the drug. Cisplatin inhibits both the JNK pathway and PTMA in a dose-dependent manner. Treatment with a JNK inhibitor also reduces PTMA protein stability and sensitizes HCC cells to cisplatin. Notably, the effects of PTMA silencing and JNK inhibition can be reversed by ectopic expression of PTMA. We show that PTMA silencing induces translocation of proapoptotic Bax to mitochondria and enhances cisplatin-induced cytochrome c release and caspase-9 activation. Conversely, ectopic expression of PTMA reverses these effects. Our results indicate that PTMA is positively regulated by JNK and protects HCC cells against cisplatin-induced cell death. The JNK/PTMA axis may thus represent a novel target for chemotherapy against HCC.

Spontaneous metastases in immunocompetent mice harboring a primary tumor driven by oncogene latent membrane protein 1 from Epstein-Barr virus.

BACKGROUND: In vitro and clinical studies suggest that the oncogene LMP1 (latent membrane protein 1) encoded by Epstein-Barr virus (EBV) plays a role in the development of nasopharyngeal carcinoma (NPC) and the formation of metastases in immunocompetent individuals. However, whether LMP1 itself is sufficient to drive these events in immunocompetent hosts remains elusive due to the lack of appropriate experimental models. The aim of this study was to study LMP1-dependent tumorigenesis and metastasis in BALB/c mice inoculated with BALB/c-3T3 cells expressing N-LMP1 (a Taiwanese NPC variant). METHODS: Following cancer cell inoculation, metastasis formation was monitored over time using PCR analysis of LMP1 as tumor marker. We also used a luciferase (Luc)-containing N-LMP1 and bioluminescent imaging (BLI) to monitor metastasis formation in a non-invasive manner. RESULTS: N-LMP1 appeared early in draining lymph nodes and in various distant organs before the rapid growth of the primary tumor. Lung metastasis was observed by BLI and further confirmed by histological examination. Furthermore, we detected luciferase signals in the lungs, even before the animals were sacrificed. CONCLUSIONS: Our results demonstrate the high metastatic character of N-LMP1 in immunocompetent hosts. Systemic tumor dissemination occurs even before aggressive tumor growth at the primary site, suggesting that early treatment of primary LMP1-associated tumors and distant micro-metastases is critical to achieve positive results.

Up-regulation of FLIP in cisplatin-selected HeLa cells causes cross-resistance to CD95/Fas death signalling.

Cisplatin-selected cervix carcinoma HeLa cell lines induced less apoptosis, and weaker activation by cisplatin or Fas-activating antibody, of mitochondrial-associated caspase-9 and death receptor-mediated caspase-8 than did parental cells. Furthermore, less DISC (death-inducing signalling complex) was formed in cisplatin-selected cell lines than in parental cells. Ac-IETD-CHO (acetyl-Ile-Glu-Thr-Asp-aldehyde), which has a certain preference for inhibiting caspase-8, or Fas-antagonistic antibody, significantly inhibited cisplatin-induced apoptosis in both parental and cisplatin-selected HeLa cell lines. These results imply that cell-surface death signalling is inducible by cisplatin; that reduction of this pathway is associated with drug resistance, and that cisplatin-selected cells acquire cross-resistance to cell-surface death signalling. Sequential up-regulation of FLIP (FLICE-like inhibitory protein), but not Bcl-2, Bcl-x(L) or inhibitors of apoptosis protein (IAPs), was observed in resistant cells but not in parental cells. The inhibition of FLIP by FLIP antisense oligonucleotides promotes cisplatin and Fas-antibody-induced apoptosis. However, the modulation of apoptosis by FLIP antisense oligonucleotides in resistant cells is greater than that in parental cells. The presented data reveal that the up-regulation of FLIP may contribute to the suppression of apoptosis and thereby change cells that are resistant to cisplatin and Fas-mediated death signals. The results also show that cancer cells that have undergone long-term chemotherapy and become chemoresistant may change the FLIP level, becoming cross-resistant to death factors such as Fas.