Significance of Plk1 regulation by miR-100 in human nasopharyngeal cancer.

Polo-like kinase 1 (Plk1) is a critical regulator of many stages of mitosis; increasing evidence indicates that Plk1 overexpression correlates with poor clinical outcome, yet its mechanism of regulation remains unknown. Hence, a detailed evaluation was undertaken of Plk1 expression in human nasopharyngeal cancer (NPC), the cellular effects of targeting Plk1 using siRNA in combination with ionizing radiation (RT) and potential upstream microRNAs (miRs) that might regulate Plk1 expression. Using immunohistochemistry, Plk1 was observed to be overexpressed in 28 of 40 (70%) primary NPC biopsies, which in turn was associated with a higher likelihood of recurrence (p = 0.018). SiPlk1 significantly inhibited Plk1 mRNA and protein expression, and decreased Cdc25c levels in NPC cell lines. This depletion resulted in cytotoxicity of C666-1 cells, enhanced by the addition of RT, mediated by G2/M arrest, increased DNA double-strand breaks, apoptosis, and caspase activation. Immunofluorescence demonstrated that the G2/M arrest was associated with aberrant spindle formation, leading to mitotic arrest. In vivo, transfection of C666-1 cells and systemic delivery of siPlk1 decreased tumour growth. MicroRNA-100 (miR-100) was predicted to target Plk1 mRNA, which was indeed underexpressed in C666-1 cells, inversely correlating with Plk1 expression. Using luciferase constructs containing the 3'-UTR of Plk1 sequence, we document that miR-100 can directly target Plk1. Hence, our data demonstrate for the first time that underexpressed miR-100 leads to Plk1 overexpression, which in turn contributes to NPC progression. Targeting Plk1 will cause mitotic catastrophe, with significant cytotoxicity both in vitro and in vivo, underscoring the important therapeutic opportunity of Plk1 in NPC.

Potential use of cetrimonium bromide as an apoptosis-promoting anticancer agent for head and neck cancer.

A potential therapeutic agent for human head and neck cancer (HNC), cetrimonium bromide (CTAB), was identified through a cell-based phenotype-driven high-throughput screen (HTS) of 2000 biologically active or clinically used compounds, followed by in vitro and in vivo characterization of its antitumor efficacy. The preliminary and secondary screens were performed on FaDu (hypopharyngeal squamous cancer) and GM05757 (primary normal fibroblasts), respectively. Potential hit compounds were further evaluated for their anticancer specificity and efficacy in combination with standard therapeutics on a panel of normal and cancer cell lines. Mechanism of action, in vivo antitumor efficacy, and potential lead compound optimizations were also investigated. In vitro, CTAB interacted additively with gamma radiation and cisplatin, two standard HNC therapeutic agents. CTAB exhibited anticancer cytotoxicity against several HNC cell lines, with minimal effects on normal fibroblasts; a selectivity that exploits cancer-specific metabolic aberrations. The central mode of cytotoxicity was mitochondria-mediated apoptosis via inhibition of H(+)-ATP synthase activity and mitochondrial membrane potential depolarization, which in turn was associated with reduced intracellular ATP levels, caspase activation, elevated sub-G(1) cell population, and chromatin condensation. In vivo, CTAB ablated tumor-forming capacity of FaDu cells and delayed growth of established tumors. Thus, using an HTS approach, CTAB was identified as a potential apoptogenic quaternary ammonium compound possessing in vitro and in vivo efficacy against HNC models.

Increased efficiency for performing colony formation assays in 96-well plates: novel applications to combination therapies and high-throughput screening.

The colony formation assay (CFA) is the gold standard for measuring the effects of cytotoxic agents on cancer cells in vitro; however, in its traditional 6-well format, it is a time-consuming assay, particularly when evaluating combination therapies. In the interest of increased efficiency, the 6-well CFA was converted to a 96-well format using an automated colony counting algorithm. The 96-well CFA was validated using ionizing radiation therapy on the FaDu (human hypopharyngeal squamous cell) and A549 (human lung) cancer cell lines. Its ability to evaluate combination therapies was investigated by the generation of dose-response curves for the combination of cisplatin and radiation therapy on FaDu and A549 cells. The 96-well CFA was then transferred to a robotic platform for evaluating its potential as a high-throughput screening (HTS) readout. The LOPAC1280 library was screened against FaDu cells, and eight putative hits were identified. Using the 96-well CFA to validate the eight putative chemicals, six of the eight were confirmed, resulting in a positive hit rate of 75%. These data indicate that the 96-well CFA can be adopted as an efficient alternative assay to the 6-well CFA in evaluating single and combination therapies in vitro, providing a possible readout that could be used on a HTS platform.

Nuclear factor-Y and Epstein Barr virus in nasopharyngeal cancer.

PURPOSE: The Epstein Barr virus (EBV) is intimately associated with nasopharyngeal cancer (NPC) in a latent state expressing a limited number of genes. The process of switching from latency to replication is not well understood, particularly in response to DNA stress; hence, the focus of this study is on an EBV-positive NPC model. EXPERIMENTAL DESIGN: C666-1 cells were exposed to radiation (2-15 Gy) or cisplatin (0.1-50 microg/mL) assayed subsequently for relative EBV copy number (BamHI) and lytic gene expression (BRLF1 and BZLF1) using quantitative real-time PCR. Chromatin immunoprecipitation was conducted to assess the interaction of the transcription factor nuclear factor-Y (NF-Y) with promoter sequences. RESULTS: Radiation-induced and cisplatin-induced BamHI expression, along with increased levels of BRLF1 and BZLF1 in a dose-dependent and time-dependent manner, associated with the immediate nuclear transactivation of the transcription factor NF-Y and its own increased transcription of NF-Y subunits 8 h posttreatment. In silico analysis revealed three putative NF-Y consensus-binding sequences in the promoter region of BRLF1, which all interacted with NF-Y in response to radiation and cisplatin, confirmed using chromatin immunoprecipitation. Introduction of dominant-negative NF-YA reduced BRLF1 expression after radiation and cisplatin by 2.8-fold; in turn, overexpression of NF-YA resulted in a 2-fold increase in both BRLF1 and BZLF1 expression. CONCLUSIONS: These results show that NF-Y is an important mediator of EBV stress response in switching from a latent to lytic state. This novel insight could provide a potential therapeutic strategy to enhance NPC response to radiation and cisplatin.

Local radiotherapy induces homing of hematopoietic stem cells to the irradiated bone marrow.

Local breast radiation therapy (RT) is associated with a 3-fold increased risk of secondary acute myeloid leukemia. As a first step in determining the mechanism(s) underlying this observation, we investigated the role of RT in mediating the active recruitment of hematopoietic stem cells (HSC) to the site of RT. Our results show in a mouse model that local RT delivered to the left leg causes preferential accumulation of bone marrow mononuclear cells to the irradiated site, with maximum signal intensity observed at 7 days post-RT. This is associated with a 4-fold higher number of donor-derived HSC present in the left leg, demonstrating recruitment of HSC to the site of RT. SDF-1, matrix metalloproteinase 2 (MMP-2), and MMP-9 expression is significantly increased in the irradiated bone marrow, and their inhibition significantly reduced HSC recruitment to the irradiated bone marrow. Our data show that local RT has significant systemic effects by recruiting HSC to the irradiated bone marrow site, a process mediated by SDF-1, MMP-2, and MMP-9. These results raise the possibility that the exposure of increased numbers of HSC at a local site to fractionated irradiation may increase the risk of leukemogenesis. Our data also suggest some opportunities for leukemia prevention in breast cancer patients undergoing RT.

Imaging and modulating antisense microdistribution in solid human xenograft tumor models.

PURPOSE: The tumor microenvironment is complex and heterogeneous, populated by tortuous irregular vasculature, hypoxic cells, and necrotic regions. These factors can all contribute to the biodistribution difficulties encountered by most cancer therapeutic agents. Antisense oligodeoxynucleotides (ASO) are a class of therapeutics where limited information is available about their distribution within a solid tumor environment. EXPERIMENTAL DESIGN: To assess ASO distribution, a fluorescein-labeled phosphorothionated ASO based on the G3139 mismatch control was injected systemically (i.v.) into tumor-bearing severe combined immunodeficient mice. Hoechst 33342 was injected i.v. to visualize active vasculature. Unstained sections were imaged through tiled fluorescence stereomicroscopy and then quantitated using novel algorithms. Tumor sections from four human tumor models were examined (CaSki, DU-145, C666-1, and C15) for hypoxia, apoptosis/necrosis, and morphology. RESULTS: For all four tumors, ASO accumulated within regions of hypoxia, necrosis, and apoptosis. Scatter plots of ASO versus active vasculature generated for each individual tumor revealed a consistent pattern of distribution of the ASO within each model. In C666-1 xenografts, the slopes of these scatter plots were significantly reduced from 0.41 to 0.16 when pretreated with the antivascular agent ZD6126 48 h before ASO injection. This was accompanied by the formation of large disseminated necrotic regions in the tumor, along with a 13.1 mmHg reduction in interstitial fluid pressure. CONCLUSIONS: These data suggest the possibility that these algorithms might offer a generalizable and objective methodology to describe the distribution of molecular therapeutic agents within a tumor microenvironment and to quantitatively assess distribution changes in response to combination therapies.

Imaging the modulation of adenoviral kinetics and biodistribution for cancer gene therapy.

To explore systemic utilization of Epstein-Barr virus (EBV)-specific transcriptionally targeted adenoviruses, three vectors were constructed to examine kinetics, specificity, and biodistribution: adv.oriP.luc, expressing luciferase under EBV-specific control; adv.SV40luc, expressing luciferase constitutively; and adv.oriP.E1A.oriP.luc, a conditionally replicating adenovirus, expressing both luciferase and E1A. Bioluminescence imaging (BLI) was conducted on tumor-bearing severe combined immunodeficient (SCID) mice (C666-1, EBV-positive human nasopharyngeal cancer) treated intravenously (i.v.) with 3 x 10(8) infectious units (ifu) of the adenoviral vectors. At 72 hours, adv.oriPluc demonstrated an 8.4-fold higher tumor signal than adv.SV40luc; adv.oriP.E1A.oriP.luc was 26.7-fold higher; however, a significant liver signal was also observed, necessitating further action to improve biodistribution. Several compounds were examined to this end, including norepinephrine, serotonin, clodronate liposomes, and STI571, to determine whether any of these measures could improve adenoviral biodistribution. Each of these interventions was assessed using BLI in mice i.v. injected with adv.oriP.luc. STI571 achieved the highest increase in tumor-to-liver ratio (TLR; 6.6-fold), which was associated with a 59% reduction in tumor interstitial fluid pressure (IFP) along with a decrease in platelet-derived growth factor-beta receptor (PDGF beta R) activation. This study reports the favorable modulation by STI571 of the biodistribution of adenoviral vectors, providing a potential approach to improving therapeutic outcome.

East-West Symposium on nasopharyngeal cancer.

BACKGROUND: To achieve greater understanding of the epidemiology, pathogenesis, molecular oncology, diagnostic, and therapeutic aspects of nasopharyngeal cancer (NPC), an international meeting was held in June 2005, Toronto, Canada. RESULTS: Further insights were obtained into the role of EBV in NPC development, with its diverse effects ranging from proliferative signals via NF-kB, to immunesuppression, to angiogenic gene regulation. Subsequently, multiple pathways are dysregulated in NPC as revealed by expression array analyses, including apoptosis, integrin, and B-catenin cascades. Advances have been made in the diagnosis and monitoring of NPC, using transoral brushings and plasma levels of EBV transcripts, which may not directly correlate with the number of circulating tumor cells, but is nevertheless informative in predicting and tracking disease response. Many novel therapies have promising results, particularly in the areas of immunotherapies, and the exploration of molecularly targeted approaches such as cetuximab or histone deacetylase inhibitors. CONCLUSIONS: The results from large randomized trials and meta-analyses have consistently demonstrated the benefit of concurrent chemotherapy with curative radiation therapy, but at a cost of greater acute and late-tissue toxicities. Further advances are required to achieve an improved understanding on the inter-relationship between environmental and genetic determinants in NPC development, to reduce the global burden of this disease. At the same time, novel therapeutic approaches are necessary to increase curability of NPC, but with reduced long-term toxicities.

Imaging the Modulation of Adenoviral Kinetics and Biodistribution for Cancer Gene Therapy.

See page 841 To explore systemic utilization of Epstein-Barr virus (EBV)-specific transcriptionally targeted adenoviruses, three vectors were constructed to examine kinetics, specificity, and biodistribution: adv.oriP.luc, expressing luciferase under EBV-specific control; adv.SV40luc, expressing luciferase constitutively; and adv.oriP.E1A.oriP.luc, a conditionally replicating adenovirus, expressing both luciferase and E1A. Bioluminescence imaging (BLI) was conducted on tumor-bearing severe combined immunodeficient (SCID) mice (C666-1, EBV-positive human nasopharyngeal cancer) treated intravenously (i.v.) with 3 × 108 infectious units (ifu) of the adenoviral vectors. At 72 hours, adv.oriPluc demonstrated an 8.4-fold higher tumor signal than adv.SV40luc; adv.oriP.E1A.oriP.luc was 26.7-fold higher; however, a significant liver signal was also observed, necessitating further action to improve biodistribution. Several compounds were examined to this end, including norepinephrine, serotonin, clodronate liposomes, and STI571, to determine whether any of these measures could improve adenoviral biodistribution. Each of these interventions was assessed using BLI in mice i.v. injected with adv.oriP.luc. STI571 achieved the highest increase in tumor-to-liver ratio (TLR; 6.6-fold), which was associated with a 59% reduction in tumor interstitial fluid pressure (IFP) along with a decrease in platelet-derived growth factor-ß receptor (PDGFßR) activation. This study reports the favorable modulation by STI571 of the biodistribution of adenoviral vectors, providing a potential approach to improving therapeutic outcome.

Gene expression profiling in cervical cancer: an exploration of intratumor heterogeneity.

PURPOSE: To explore intratumor heterogeneity in gene expression profiles from patients with cervical cancer. EXPERIMENTAL DESIGN: A total of 33 biopsies were obtained from 11 patients, sampling between two and five different areas for each tumor. The extracted RNA was hybridized onto the Affymetrix U133 Plus 2.0 oligonucleotide chip. The variance of expression within a patient (W), between patients (B) and the total variance (T = W + B) were calculated for each ProbeSet, and the ratio W/T was used as a measure of intratumor heterogeneity. Gene Ontology functional analysis was done to assess the function of genes that had high W/T (top 10%) and low W/T (bottom 10%) values. RESULTS: In total, 448 ProbeSets (2.2% of the total) had W/T < 0.10, indicating low intratumor heterogeneity, and 537 ProbeSets (2.7% of the total) had W/T > 0.90, indicating high intratumor heterogeneity. In total 14,473 ProbeSets (72.4%) had higher intertumor than intratumor heterogeneity (W/T < 0.5). Genes with low intratumor heterogeneity were characterized by a statistically significant enrichment of immune-related functions (P < 0.0001). Genes with high intratumor heterogeneity were characterized by a significant tendency towards nuclear localization and nucleic acid binding (both P < 0.0001). For genes with W/T > 0.5, more than six biopsies would be required to minimize the intratumoral heterogeneity to <0.15; if W/T is 0.3 to 0.4, four biopsies are required; and for low W/T of 0.16 to 0.3, only two to three biopsies would be needed. CONCLUSION: Although the intratumor heterogeneity was low for the majority of the tested ProbeSets, for many genes, multiple biopsies are required to obtain a reliable estimate of gene expression.

Prognostic significance of the Epstein-Barr virus, p53, Bcl-2, and survivin in nasopharyngeal cancer.

PURPOSE: Nasopharyngeal cancer (NPC) is a malignant epithelial carcinoma which is intimately associated with EBV. The latent presence of EBV affects the function of p53, Bcl-2, and survivin. We thus investigated the relationship between EBV status, p53, Bcl-2, and survivin in biopsy specimens from patients with primary NPC. EXPERIMENTAL DESIGN: Archival formalin-fixed, paraffin-embedded NPC biopsies were evaluated in 80 patients treated with curative radiation from a single institution. The presence of EBV was determined using EBER in situ hybridization, whereas p53, Bcl-2, and survivin were assessed using immunohistochemistry. RESULTS: The majority of NPC specimens in this patient cohort were EBER-positive (64 of 78, or 82%), which in turn, was significantly associated with ethnicity (P = 0.0007), and WHO subtype 2A/2B (P = 0.04). EBER-positive tumors were also associated with p53 (P = 0.002), Bcl-2 (P = 0.04), and nuclear survivin (P = 0.03) expression. Patients with EBER-positive NPC fared better, with a 10-year overall survival of 68% versus 48% for EBER-negative patients (P = 0.03). For nuclear survivin, patients with either low or high nuclear survivin fared worse than patients with intermediate survivin expression (P = 0.05), suggesting that there is an optimal proportion of survivin-expressing cells for best function and clinical outcome. CONCLUSIONS: With an extended median follow-up time of 11.4 years, EBV status remains a strong predictor for overall survival in NPC. EBV-positive NPC has strong molecular associations with p53, Bcl-2, and survivin expression. Furthermore, we provide clinical data revealing the potentially dual nature of survivin in predicting clinical outcome.

Potential use of alexidine dihydrochloride as an apoptosis-promoting anticancer agent.

Despite advances in surgery, radiation, and chemotherapy, novel therapeutics are needed for head and neck cancer treatment. The objective of this current study was to evaluate alexidine dihydrochloride as a novel compound lead for head and neck cancers. Using a tetrazolium-based assay, the dose required to reduce cell viability by 50% (ED50) was found to be approximately 1.8 micromol/L in FaDu (human hypopharyngeal squamous cancer) and approximately 2.6 micromol/L in C666-1 (human undifferentiated nasopharyngeal cancer) cells. In contrast, the ED50 values were much higher in untransformed cells, specifically at approximately 8.8 micromol/L in GM05757 (primary normal human fibroblast), approximately 8.9 micromol/L in HNEpC (primary normal human nasal epithelial), and approximately 19.6 micromol/L in NIH/3T3 (mouse embryonic fibroblast) cells. Alexidine dihydrochloride did not interfere with the activities of cisplatin, 5-fluorouracil, or radiation, and interacted in a less-than-additive manner. DNA content analyses and Hoechst 33342 staining revealed that this compound induced apoptosis. Alexidine dihydrochloride-induced mitochondrial damage was visualized using transmission electron microscopy. Mitochondrial membrane potential (DeltaPsiM) depolarization was detectable after only 3 hours of treatment, and was followed by cytosolic Ca2+ increase along with loss of membrane integrity/cell death. Caspase-2 and caspase-9 activities were detectable at 12 hours, caspase-8 at 24 hours, and caspase-3 at 48 hours. FaDu cell clonogenic survival was reduced to < 5% with 1 micromol/L alexidine dihydrochloride, and, correspondingly, this compound decreased the in vivo tumor-forming potential of FaDu cells. Thus, we have identified alexidine dihydrochloride as the first bisbiguanide compound with anticancer specificity.

Benzethonium chloride: a novel anticancer agent identified by using a cell-based small-molecule screen.

PURPOSE: This study aims to identify a novel therapeutic agent for head and neck cancer and to evaluate its antitumor efficacy. EXPERIMENTAL DESIGN: A cell-based and phenotype-driven high-throughput screening of approximately 2,400 biologically active or clinically used compounds was done using a tetrazolium-based assay on FaDu (hypopharyngeal squamous cancer) and NIH 3T3 (untransformed mouse embryonic fibroblast) cells, with secondary screening done on C666-1 (nasopharyngeal cancer) and GM05757 (primary normal human fibroblast) lines. The "hit" compound was assayed for efficacy in combination with standard therapeutics on a panel of human cancer cell lines. Furthermore, its mode of action (using transmission electron microscopy and flow cytometry) and its in vivo efficacy (using xenograft models) were evaluated. RESULTS: Benzethonium chloride was identified as a novel cancer-specific compound. For benzethonium (48-hour incubation), the dose required to reduce cell viability by 50% was 3.8 micromol/L in FaDu, 42.2 micromol/L in NIH 3T3, 5.3 micromol/L in C666-1, and 17.0 micromol/L in GM05757. In vitro, this compound did not interfere with the effects of cisplatin, 5-fluorouracil, or gamma-irradiation. Benzethonium chloride induced apoptosis and activated caspases after 12 hours. Loss of mitochondrial membrane potential (DeltaPsiM) preceded cytosolic Ca2+ increase and cell death. In vivo, benzethonium chloride ablated the tumor-forming ability of FaDu cells, delayed the growth of xenograft tumors, and combined additively with local tumor radiation therapy. Evaluation of benzethonium chloride on the National Cancer Institute/NIH Developmental Therapeutics Program 60 human cancer cell lines revealed broad-range antitumor activity. CONCLUSIONS: This high-throughput screening identified a novel antimicrobial compound with significant broad-spectrum anticancer activity.

Multiple dysregulated pathways in nasopharyngeal carcinoma revealed by gene expression profiling.

Gene expression profiling was conducted using primary human nasopharyngeal carcinoma (NPC) biopsy samples to improve the understanding of the molecular pathways defining NPC and to identify novel potential therapeutic targets. RNA samples were extracted from 36 patients suspected to have NPC and hybridized onto the Affymetrix U133A chip. NPC was diagnosed in 19 patients, 11 had lymphoid hyperplasia (LH), and 6 were "normal" biopsies. Clinical stages for these NPC patients ranged from I-IV, including one M1. All NPC patients (except the M1) were treated with curative intent, which included radiotherapy alone (4 patients), or combined with chemotherapy (14 patients). Unsupervised clustering demonstrated a distinct NPC expression pattern, compared to normal biopsies. Subsequent Significance Analysis of Microarrays (SAM) derived from 14 NPC and 6 normal samples discovered 1,089 differentially regulated genes. Pathway analyses revealed novel insights into the mechanisms leading to NPC, whereby upregulation of NFkappaB2 and survivin play central roles in increasing resistance to apoptosis, and changes in integrin and WNT/beta-catenin signaling leading to uncontrolled proliferation. The role of survivin in resisting apoptosis in NPC was confirmed by RNA interference. Our data provide novel insights into the development and progression of NPC, and suggest survivin as a novel therapeutic target for NPC.

Combination bcl-2 antisense and radiation therapy for nasopharyngeal cancer.

PURPOSE: A wide variety of tumors depend on the dysregulation of Bcl-2 family proteins for survival. The resulting apoptotic block can often provide a mechanism for resistance to anticancer treatments, such as chemotherapy and radiation. This current study evaluates the efficacy of combining systemically delivered Bcl-2 phosphorothioate antisense (Bcl-2 ASO) and radiation for nasopharyngeal cancer therapy. RESULTS: Antisense uptake was unaffected by 0, 3, or 6 Gy radiation. Radiation decreased the fraction of viable C666-1 cells to 60%, with a further decrease to 40% in combination with Bcl-2 ASO. Despite a modest in vitro effect, Bcl-2 ASO alone caused the regression of established xenograft tumors in mice, extending survival by 15 days in a C666-1 and by 6 days in a C15 model. The survival times for mice treated with both Bcl-2 ASO and radiation increased by 52 days in C666-1 and by 20 days in C15 tumors. This combination resulted in a more-than-additive effect in C666-1 tumors. Less impressive gains observed in C15 tumors might be attributable to higher expression of antiapoptotic Bcl-2 family proteins and limited drug distribution in the tumor. Retreatment of C666-1 tumors with the Bcl-2 ASO-radiation combination, however, was effective, resulting in mice surviving for >80 days relative to untreated controls. CONCLUSIONS: Our results show that the Bcl-2 ASO and radiation combination is a highly potent therapy for nasopharyngeal cancer. Further examination of combination therapy with radiation and other Bcl-2 family-targeted anticancer agents in both preclinical and clinical settings is definitely warranted.

A novel muscle-specific enhancer identified within the deletion overlap region of two XLDC patients lacking muscle exon 1 of the human dystrophin gene.

Previous studies point to the involvement of several discrete transcriptional enhancers in the modulation of dystrophin gene expression in skeletal and cardiac muscle. Analysis of deletion breakpoints in two X-linked dilated cardiomyopathy patients with mutations that remove muscle exon 1 identified a 3.2-kb deletion overlap region (XLDC3.2) between -1199 and +2057 bp predicted to contain regulatory elements essential for dystrophin gene expression in cardiac muscle. A novel-sequence-based search strategy was used to identify a 543-bp region downstream of muscle exon 1 rich in cardiac-specific transcriptional elements. Designated dystrophin muscle enhancer 2 (DME2), this candidate enhancer was seen to function in a position- and orientation-independent manner in muscle cell lines but not in fibroblasts. As only modest activity was observed in primary neonatal rat cardiomyocytes, DME2 is thought to play a role in dystrophin gene regulation at later stages of cardiac muscle development.