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.

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.

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.

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.

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.

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.

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.

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.

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.

Identification of the ß-catenin/JNK/prothymosin-alpha axis as a novel target of sorafenib in hepatocellular carcinoma cells.

Sorafenib is a kinase inhibitor used as anticancer drug against various human tumors, including advanced hepatocellular carcinoma (HCC). ß-Catenin and prothymosin alpha (PTMA) are overexpressed in HCC and other tumors. Previous studies have shown that PTMA expression modulates the response of HCC cells to sorafenib. However, the underlying mechanism of PTMA activity in this context remains unclear. We show here that sorafenib inhibits both ß-catenin and PTMA in a dose-dependent manner. Silencing ß-catenin reduces PTMA level and sensitizes HCC cells to sorafenib. In contrast, ectopic expression of ß-catenin induces PTMA expression and cell resistance to the drug. Sorafenib inhibits PTMA expression at the transcriptional level by inhibiting the ß-catenin pathway. Nucleotide deletion analysis of the PTMA gene promoter reveals that a DNA segment lying 1,500-1,600 bp upstream of the PTMA transcription start site represents an AP-1-binding site that is critical for ß-catenin modulation of gene transcription in response to sorafenib. In addition, chemical inhibitors that target JNK abrogate ß-catenin/AP-1 binding to the endogenous PTMA gene and reduces PTMA transcription and protein expression. Silencing of ß-catenin or c-Fos induces similar effects on gene regulation and these are reversed by ectopic expression of ß-catenin. Mutations in the PTMA promoter at the predicted ß-catenin/AP-1 binding site partly abrogate sorafenib's effects on PTMA transcription. These results indicate that PTMA is induced by the oncoprotein ß-catenin and protects HCC cells against sorafenib-induced cell death. The ß-catenin/JNK/PTMA axis may thus represent a novel target for chemotherapy against HCC.

Growth-Arrest-Specific 7 Gene Regulates Neural Crest Formation and Craniofacial Development in Zebrafish.

Growth-arrest-specific 7 (Gas7) is preferentially expressed in the nervous system and plays an important role during neuritogenesis in vertebrates. We recently demonstrated that gas7 is highly expressed in zebrafish neurons, where it regulates neural development. The possibility that gas7 may also regulate the development of other tissues remains to be examined. In this study, we investigate the role of Gas7 in the development of craniofacial tissues. Knockdown of gas7 using morpholino oligomers produced abnormal phenotypes in neural crest (NC) cells and their derivatives. NC-derived cartilage maturation was altered in Gas7 morphants as revealed by aberrant sox9b and dlx2 expression, a phenotype that could be rescued by coinjection of gas7 mRNA. While rhombomere morphology remained normal in Gas7 morphants, we observed reduced expression of the prechondrogenic genes sox9b and dlx2 in cells populating the posterior pharyngeal arches, but the fundamental structure of pharyngeal arches was preserved. In addition, NC cell sublineages that migrate to form neurons, glial cells, and melanocytes were altered in Gas7 morphants as revealed by aberrant expression of neurod, foxd3, and mitfa, respectively. Development of NC progenitors was also examined in Gas7 morphants at 12 hpf, and we observed that the reduction of cell precursors in Gas7 morphants was due to increased apoptosis level. These results indicate that the formation of NC progenitors and derivatives depends on Gas7 expression. Our observations also suggest that Gas7 regulates the formation of NC derivatives constituting the internal tissues of pharyngeal arches, without affecting the fundamental structure of mesodermal-derived pharyngeal arches.

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.

Integrative transcriptomics-based identification of cryptic drivers of taxol-resistance genes in ovarian carcinoma cells: Analysis of the androgen receptor.

A systematic analysis of the genes involved in taxol resistance (txr) has never been performed. In the present study, we created txr ovarian carcinoma cell lines to identify the genes involved in chemoresistance. Transcriptome analysis revealed 1,194 overexpressed genes in txr cells. Among the upregulated genes, more than 12 cryptic transcription factors were identified using MetaCore analysis (including AR, C/EBPß, ERα, HNF4α, c-Jun/AP-1, c-Myc, and SP-1). Notably, individual silencing of these transcription factors (except HNF4`)sensitized txr cells to taxol. The androgen receptor (AR) and its target genes were selected for further analysis. Silencing AR using RNA interference produced a 3-fold sensitization to taxol in txr cells, a response similar to that produced by silencing abcb1. AR silencing also downregulated the expression of prominent txr gene candidates (including abcb1, abcb6, abcg2, bmp5, fat3, fgfr2, h1f0, srcrb4d, and tmprss15). In contrast, AR activation using the agonist DHT upregulated expression of the target genes. Individually silencing seven out of nine (78%) AR-regulated txr genes sensitized txr cells to taxol. Inhibition of AKT and JNK cellular kinases using chemical inhibitors caused a dramatic suppression of AR expression. These results indicate that the AR represents a critical driver of gene expression involved in txr.

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.

Silencing of Taxol-Sensitizer Genes in Cancer Cells: Lack of Sensitization Effects.

A previous genome-wide screening analysis identified a panel of genes that sensitize the human non-small-cell lung carcinoma cell line NCI-H1155 to taxol. However, whether the identified genes sensitize other cancer cells to taxol has not been examined. Here, we silenced the taxol-sensitizer genes identified (acrbp, atp6v0d2, fgd4, hs6st2, psma6, and tubgcp2) in nine other cancer cell types (including lung, cervical, ovarian, and hepatocellular carcinoma cell lines) that showed reduced cell viability in the presence of a sub-lethal concentration of taxol. Surprisingly, none of the genes studied increased sensitivity to taxol in the tested panel of cell lines. As observed in H1155 cells, SKOV3 cells displayed induction of five of the six genes studied in response to a cell killing dose of taxol. The other cell types were much less responsive to taxol. Notably, four of the five inducible taxol-sensitizer genes tested (acrbp, atp6v0d2, psma6, and tubgcp2) were upregulated in a taxol-resistant ovarian cancer cell line. These results indicate that the previously identified taxol-sensitizer loci are not conserved genetic targets involved in inhibiting cell proliferation in response to taxol. Our findings also suggest that regulation of taxol-sensitizer genes by taxol may be critical for acquired cell resistance to the drug.

Oncogenic c-Myc and prothymosin-alpha protect hepatocellular carcinoma cells against sorafenib-induced apoptosis.

Prothymosin alpha (PTMA) is overexpressed in various human tumors, including hepatocellular carcinoma (HCC). The significance of PTMA overexpression and its underlying mechanism remain unclear. We show here that silencing PTMA sensitizes HCC cells to the kinase inhibitor sorafenib. In contrast, ectopic expression of PTMA induces cell resistance to the drug. While inhibitors targeting JNK, ERK or PI3K reduce PTMA expression, only ERK activation is suppressed by sorafenib. In addition, inhibition of ERK produces a dramatic decrease in both endogenous PTMA level and promoter activation. Ectopic expression of active MKK1/2 considerably induces PTMA expression. We also identify a sorafenib-responsive segment lying 1000-1500-bp upstream of the PTMA transcription start site and observe that it is controlled by c-Myc and ERK. Mutation in the PTMA promoter at the predicted c-Myc binding site and silencing of c-Myc both abrogate sorafenib's effect on PTMA transcription. We also find that silencing PTMA potentiates Bax translocation to mitochondria in response to sorafenib and this is associated with increased cytochrome c release from mitochondria and enhanced caspase-9 activation. These results indicate that PTMA is positively regulated by the oncoprotein c-Myc and protects HCC cells against sorafenib-induced cell death, thus identifying PTMA as a new target for chemotherapy against HCC.

Mechanisms of p53 degradation.

The tumor suppressor p53 plays various functional roles in the cell by regulating multiple regulatory signals that ensure adequate temporal and spatial responses to cellular stress. p53 is usually kept inactive due to ubiquitination by a number of E3 ubiquitin ligases that target p53 for proteasomal degradation. The ubiquitously expressed proto-oncogene Mdm2 is the major E3 ubiquitin ligase involved in this process and is critical for regulating p53 homeostasis. Ubiquitination by E3 ligases may induce cellular relocation of p53 and determine the outcome of p53-mediated stress response, including cell proliferation, apoptosis and efficacy of cancer therapy. In addition to marking p53 for proteasomal degradation, ubiquitination acts as a signal for the degradation-independent functions of p53, such as nuclear export and transcriptional activation. Importantly, the reversible nature of the ubiquitination process and the identification of de-ubiquitination enzymes acting on p53 have added yet another layer of regulatory mechanism controlling p53 activity. This review highlights our current understanding of the mechanisms underlying p53 degradation as well as the significance of the p53 pathway in response to genotoxic stress.

Transcriptomic profiling of taxol-resistant ovarian cancer cells identifies FKBP5 and the androgen receptor as critical markers of chemotherapeutic response.

Taxol is a mitotoxin widely used to treat human cancers, including of the breast and ovary. However, taxol resistance (txr) limits treatment efficacy in human patients. To study chemoresistance in ovarian cancer, we established txr ovarian carcinoma cells derived from the SKOV3 cell lineage. The cells obtained were cross-resistant to other mitotoxins such as vincristine while they showed no resistance to the genotoxin cisplatin. Transcriptomic analysis identified 112 highly up-regulated genes in txr cells. Surprisingly, FK506-binding protein 5 (FKBP5) was transiently up-regulated 100-fold in txr cells but showed decreased expression in prolonged culture. Silencing of FKBP5 sensitized txr cells to taxol, whereas ectopic expression of FKBP5 increased resistance to the drug. Modulation of FKBP5 expression produced similar effects in response to vincristine but not to cisplatin. We observed that a panel of newly identified txr genes was trancriptionally regulated by FKBP5 and silencing of these genes sensitized cells to taxol. Notably, immunoprecipitation experiments revealed that FKBP5 forms a protein complex with the androgen receptor (AR), and this complex regulates the transcriptional activity of both proteins. Furthermore, we found that the Akt kinase pathway is regulated by FKBP5. These results indicate that the FKBP5/AR complex may affect cancer cell sensitivity to taxol by regulating expression of txr genes. Our findings suggest that mitotoxin-based treatment against ovarian cancer should be avoided when the Akt/FKBP5/AR axis is activated.

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.