Emma Ito

Nasopharyngeal carcinoma: The next challenges

Nasopharyngeal carcinoma (NPC) differs from other head and neck cancers in its aetiology, epidemiology and potential therapeutic options. Despite cure for the majority of the patients, challenges still exist in the prevention of disease relapse, treatment of patients with refractory or metastatic NPC and the management of long-term toxicities. This article discusses the specific challenges in pushing the boundaries of NPC treatments further, with an emphasis on prognostic/predictive markers, molecularly targeted therapies, immunotherapies and the areas of interest with regard to long-term toxicities arising from therapeutic interventions.

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.

Micro-RNAs as diagnostic or prognostic markers in human epithelial malignancies

Micro-RNAs (miRs) are important regulators of mRNA and protein expression; the ability of miR expression profilings to distinguish different cancer types and classify their sub-types has been well-described. They also represent a novel biological entity with potential value as tumour biomarkers, which can improve diagnosis, prognosis, and monitoring of treatment response for human cancers. This endeavour has been greatly facilitated by the stability of miRs in formalin-fixed paraffin-embedded (FFPE) tissues, and their detection in circulation. This review will summarize some of the key dysregulated miRs described to date in human epithelial malignancies, and their potential value as molecular bio-markers in FFPE tissues and blood samples. There remain many challenges in this domain, however, with the evolution of different platforms, the complexities of normalizing miR profiling data, and the importance of evaluating sufficiently-powered training and validation cohorts. Nonetheless, well-conducted miR profiling studies should contribute important insights into the molecular aberrations driving human cancer development and progression.

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 γ 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-G1 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.

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 ∼1.8 μmol/L in FaDu (human hypopharyngeal squamous cancer) and ∼2.6 μmol/L in C666-1 (human undifferentiated nasopharyngeal cancer) cells. In contrast, the ED50 values were much higher in untransformed cells, specifically at ∼8.8 μmol/L in GM05757 (primary normal human fibroblast), ∼8.9 μmol/L in HNEpC (primary normal human nasal epithelial), and ∼19.6 μmol/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 (ΔΨM) 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 μmol/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. [Mol Cancer Ther 2006;5(9):2234–40]

On the Path to Seeking Novel Radiosensitizers

Radiation therapy is a highly effective cancer treatment modality, and extensive investigations have been undertaken over the years to augment its efficacy in the clinic. This review summarizes the current understanding of the biologic bases underpinning many of the clinically used radiosensitizers. In addition, this review illustrates how the advent of innovative, high-throughput technologies with integration of different disciplines could be harnessed for an expeditious discovery process for novel radiosensitizers, providing an exciting future for such pursuits in radiation biology and oncology.

TARGETING POLO-LIKE KINASE 1 ENHANCES RADIATION EFFICACY FOR HEAD-AND-NECK SQUAMOUS CELL CARCINOMA

PURPOSE To investigate the efficacy of targeting polo-like kinase 1 (Plk1) combined with ionizing radiotherapy (RT) for head-and-neck squamous cell carcinoma (HNSCC). METHODS AND MATERIALS Polo-like kinase 1 messenger ribonucleic acid (mRNA) was targeted by small interfering RNA (siRNA) transfection into the FaDu HNSCC cell line; reduction was confirmed using quantitative real-time polymerase chain reaction. The cellular effects were assessed using [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium], clonogenic, flow cytometric, and caspase assays. In vivo efficacy of siPlk1 was evaluated using mouse xenograft models. RESULTS Small interfering Plk1 significantly decreased Plk1 mRNA expression, while also increasing cyclin B1 and p21(Waf1/CIP1) mRNA levels after 24 h. This depletion resulted in a time-dependent increase in FaDu cytotoxicity, which was enhanced by the addition of RT. Flow cytometric and caspase assays demonstrated progressive apoptosis, DNA double-strand breaks (gamma-H2AX), G2/M arrest, and activation of caspases 3 and 7. Implantation of siPlk1-treated FaDu cells in severe combined immunodeficient mice delayed tumor formation, and systemic administration of siPlk1 inhibited tumor growth enhanced by RT. CONCLUSIONS These data demonstrate the suitability of Plk1 as a potential therapeutic target for HNSCC, because Plk1 depletion resulted in significant cytotoxicity in vitro and abrogated tumor-forming potential in vivo. The effects of Plk1 depletion were enhanced with the addition of RT, indicating that Plk1 represents an important potential radiation sensitizer for HNSCC.

Therapeutic Efficacy of Seliciclib in Combination with Ionizing Radiation for Human Nasopharyngeal Carcinoma

Purpose: Seliciclib is a small-molecule cyclin-dependent kinase inhibitor, which has been reported to induce apoptosis and cell cycle arrest in EBV-negative nasopharyngeal carcinoma cell lines. Because most nasopharyngeal carcinoma patients harbor EBV, we proceeded to evaluate the cytotoxic effects of seliciclib in EBV-positive nasopharyngeal carcinoma models. Experimental Design: Cytotoxicity of seliciclib was investigated in the EBV-positive cell line C666-1 and the C666-1 and C15 xenograft models. Caspase activities and cell cycle analyses were measured by flow cytometry. Efficacy of combined treatment of seliciclib with radiation therapy was also evaluated. Results: Seliciclib caused significant cytotoxicity in the C666-1 cells in a time- and dose-dependent manner, with accumulation of cells in both sub-G1 and G2-M phases, indicative of apoptosis and cell cycle arrest, respectively. Caspase-2, -3, -8, and -9 activities were all increased, with caspase-3 being the most significantly activated at 48 h after treatment. These cells also showed a reduction of Mcl-1 mRNA and protein levels. Combined treatment of seliciclib with radiation therapy showed a synergistic interaction with enhanced cytotoxicity in C666-1 cells and delayed repair of double-strand DNA breaks. For in vivo models, significant delays in tumor growth were observed for both C666-1 and C15 tumors, which were associated with enhanced apoptosis as determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and immunohistochemistry analyses. Conclusions: Seliciclib enhanced the antitumor efficacy of radiation therapy in EBV-positive nasopharyngeal carcinoma, characterized by G2-M arrest, and apoptosis, associated with an induction in caspase activity. This process is mediated by reduction in Mcl-1 expression and by attenuation of double-strand DNA break repair.

Nasopharyngeal Cancer: Molecular Landscape

Nasopharyngeal carcinoma (NPC) is a unique epithelial malignancy arising from the superior aspect of the pharyngeal mucosal space, associated with latent Epstein-Barr virus infection in most cases. The capacity to characterize cancer genomes in unprecedented detail is now providing insights into the genesis and molecular underpinnings of this disease. Herein, we provide an overview of the molecular aberrations that likely drive nasopharyngeal tumor development and progression. The contributions of major Epstein-Barr virus-encoded factors, including proteins, small RNAs, and microRNAs, along with their interactions with pathways regulating cell proliferation and survival are highlighted. We review recent analyses that clearly define the role of genetic and epigenetic variations affecting the human genome in NPC. These findings point to the impact of DNA methylation and histone modifications on gene expression programs that promote this malignancy. The molecular interactions that allow NPC cells to evade immune recognition and elimination, which is crucial for the survival of cells expressing potentially immunogenic viral proteins, are also described. Finally, the potential utility of detecting host and viral factors for the diagnosis and prognosis of NPC is discussed. Altogether, the studies summarized herein have greatly expanded our knowledge of the molecular biology of NPC, yet much remains to be uncovered. Emerging techniques for using and analyzing well-annotated biospecimens from patients with NPC will ultimately lead to a greater level of understanding, and enable improvements in precision therapies and clinical outcomes.

Efficacy of Combining GMX1777 with Radiation Therapy for Human Head and Neck Carcinoma

Purpose: Rapidly metabolizing tumor cells have elevated levels of nicotinamide phosphoribosyltransferase, an enzyme involved in NAD+ biosynthesis, which serves as an important substrate for proteins involved in DNA repair. GMX1777, which inhibits nicotinamide phosphoribosyltransferase, was evaluated in two human head and neck cancer models in combination with radiotherapy. Experimental Design: Effects of GMX1777-mediated radiosensitization were examined via metabolic and cytotoxicity assays in vitro; mechanism of action, in vivo antitumor efficacy, and radiosensitization were also investigated. Results: IC50 values of GMX1777 for FaDu and C666-1 cells were 10 and 5 nmol/L, respectively, which interacted synergistically with radiotherapy. GMX1777 induced a rapid decline in intracellular NAD+ followed by ATP reduction associated with significant cytotoxicity. These metabolic changes were slightly increased with the addition of radiotherapy, although poly(ADP-ribose) polymerase activity was significantly reduced when GMX1777 was combined with radiotherapy, thereby accounting for the synergistic cytotoxicity of these two modalities. Systemic GMX1777 administration with local tumor radiotherapy caused complete disappearance of FaDu and C666-1 tumors for 50 and 20 days, respectively. There was also significant reduction in tumor vascularity, particularly for the more sensitive FaDu model. [18F]FDG-positron emission tomography/computed tomography images showed reduction in [18F]FDG uptake after GMX1777 administration, showing decreased glucose metabolism in vivo. Conclusions: Our data represent the first report showing that GMX1777 plus radiotherapy is an effective therapeutic strategy for head and neck cancer, mediated via pleiotropic effects of inhibition of DNA repair and tumor angiogenesis, while sparing normal tissues. Therefore, GMX1777 combined with radiotherapy definitely warrants clinical evaluation in human head and neck cancer patients. Clin Cancer Res; 16(3); 898–911