Advanced technologies for studying circulating tumor cells at the protein level.

Metastasis is the main cause of cancer death. As the tumor progresses, cells from the primary tumor site are shed into the bloodstream as circulating tumor cells (CTCs). Eventually, these cells colonize other organs and form distant metastases. It is therefore imperative that we gain a better understanding of the biological characteristics of CTCs for development of novel treatment modalities to minimize metastasis-associated cancer deaths. In recent years, rapid developments in technologies for the study of CTCs have taken place. We now have a variety of tools for the isolation and examination of CTCs which were not available before. This review introduces some commonly used protein markers in CTC investigations and summarizes a few advanced technologies which have been successfully applied for studying CTC biology at the protein level.

CXCR6 and CCR5 localize T lymphocyte subsets in nasopharyngeal carcinoma.

The substantial T lymphocyte infiltrate found in cases of nasopharyngeal carcinoma (NPC) has been implicated in the promotion of both tumor growth and immune escape. Conversely, because malignant NPC cells harbor the Epstein-Barr virus, this tumor is a candidate for virus-specific T cell-based therapies. Preventing the accumulation of tumor-promoting T cells or enhancing the recruitment of tumor-specific cytotoxic T cells offers therapeutic potential. However, the mechanisms involved in T cell recruitment to this tumor are poorly understood. Comparing memory T cell subsets that have naturally infiltrated NPC tissue with their counterparts from matched blood revealed enrichment of CD8(+), CD4(+), and regulatory T cells expressing the chemokine receptor CXCR6 in tumor tissue. CD8(+) and (nonregulatory) CD4(+) T cells also were more frequently CCR5(+) in tumor than in blood. Ex vivo studies demonstrated that both receptors were functional. CXCL16 and CCL4, unique chemokine ligands for CXCR6 and CCR5, respectively, were expressed by the malignant cells in tumor tissue from the majority of NPC cases, as was another CCR5 ligand, CCL5. The strongest expression of CXCL16 was found on tumor-infiltrating cells. CCL4 was detected on the tumor vasculature in a majority of cases. These findings suggest that CXCR6 and CCR5 play important roles in T cell recruitment and/or retention in NPC and have implications for the pathogenesis and treatment of this tumor.

K252a induces anoikis-sensitization with suppression of cellular migration in Epstein-Barr virus (EBV)--associated nasopharyngeal carcinoma cells.

Recent studies revealed an unexpected role of the neurotrophin receptor pathway, BDNF/TrkB signaling, in cancer metastasis and anoikis (i.e. detachment-induced cell death). Survival of cancer cells in detached state (known as anoikis-resistance) is known to be pre-requisite for metastasis. Nasopharyngeal carcinoma (NPC), an endemic head and neck cancer in Southeast Asia, is highly invasive, metastatic, and etiologically associated with Epstein-Barr virus (EBV, an oncovirus) infection. Mechanistic studies on the invasive/metastatic nature of NPC can facilitate the development of anti-metastatic therapy in NPC. Thus far, the role of BDNF/TrkB signaling in virus-associated human cancer is unclear. Here, using multiple cell line models of NPC with EBV-association (HONE-1-EBV, HK1-LMP1 and C666-1), we investigated the potential involvement of BDNF/TrkB signaling in cellular migration and anoikis-resistant characteristics of NPC. We found that all three EBV-associated NPC cell lines tested were intrinsically anoikis-resistant (i.e. survived in detached state) and expressed both BDNF and TrkB. BDNF stimulation induced cellular migration, but not proliferation of these cells. Further, we examined if pharmacologic targeting of anoikis-resistance of NPC cells can be achievable by a proof-of-concept Trk inhibitor, K252a, in these EBV-associated NPC models. Our results demonstrated that K252a, was able to attenuate BDNF-induced migration and proliferation of NPC cells. More importantly, we demonstrated for the first time that K252a harbored potent anoikis-sensitization activity (i.e. sensitizing cancer cells to detachment-induced cell death) against EBV-associated human cancer cells, namely NPC cells. This proof-of-concept study demonstrated that K252a, a Trk inhibitor, can potentially be used as an anoikis-sensitizing agent in NPC.

Hypoxia-targeting by tirapazamine (TPZ) induces preferential growth inhibition of nasopharyngeal carcinoma cells with Chk1/2 activation.

Hypoxia is commonly developed in solid tumors, which contributes to metastasis as well as radio- and chemo-resistance. Nasopharyngeal carcinoma (NPC) is a highly invasive and metastatic head and neck cancer prevalent in Southeast Asia with a high incidence rate of 15-30/100,000 persons/year (comparable to that of pancreatic cancer in the US). Previous clinical studies in NPC showed that hypoxia is detected in almost 100% of primary tumors and overexpression of hypoxia markers correlated with poor clinical outcome. Tirapazamine (TPZ) is a synthetic hypoxia-activated prodrug, which preferentially forms cytotoxic and DNA-damaging free radicals under hypoxia, thus selectively eradicate hypoxic cells. Here, we hypothesized that specific hypoxia-targeting by this clinical trial agent may be therapeutic for NPC. Our findings demonstrated that under hypoxia, TPZ was able to induce preferential growth inhibition of NPC cells, which was associated with marked cell cycle arrest at S-phase and PARP cleavage (a hallmark of apoptosis). Examination of S-phase checkpoint regulators revealed that Chk1 and Chk2 were selectively activated by TPZ in NPC cells under hypoxia. Hypoxia-selectivity of TPZ was also demonstrated by preferential downregulation of several important hypoxia-induced markers (HIF-1α, CA IX and VEGF) under hypoxia. Furthermore, we demonstrated that TPZ was equally effective and hypoxia-selective even in the presence of the EBV oncoprotein, LMP1 or the EBV genome. In summary, encouraging results from this proof-of-concept study implicate the therapeutic potential of hypoxia-targeting approaches for the treatment of NPC.

Novel therapeutic target for head and neck squamous cell carcinoma: HGF-MET signaling pathway.

Head and neck squamous cell carcinoma (HNSCC) represents a devastating type of malignancy characterized by its high incidence of regional and distant metastases at the time of diagnosis. Vital physiological functions in the upper aerodigestive tract are often impaired as a result of the disease and treatment for the disease, giving rise to severe morbidity in patients suffering from this type of cancer. It is crucial to delineate the aberrant growth signaling pathways in HNSCC cells and develop specific target therapies for the disease to improve the treatment outcome. Although the epidermal growth factor receptor pathway has been extensively studied in HNSCC and anti-epidermal growth factor receptor therapy has already shown promise in treating HNSCC in phase III clinical trials, the signaling pathway that accounts for the highly invasive phenotype of HNSCC needs to be defined and also therapeutically targeted. The hepatocyte growth factor-MET signaling pathway has been studied extensively over the past two decades and it is now clear that it plays an important role in mediating invasive growth of many types of cancer. Here, we review comprehensively the evidence on hepatocyte growth factor-MET cascade being a key in the signaling pathway in mediating invasive growth of HNSCC and the potential of this signaling pathway to be a therapeutic target for the treatment of HNSCC.

Human papillomavirus DNA detection in menstrual blood from patients with cervical intraepithelial neoplasia and condyloma acuminatum.

The Papanicolaou test generates pain and embarrassment, and cytology screening has limited sensitivity for detection of cervical neoplasia. These factors urge the use of another screening test that can overcome these limitations. We explore a completely noninvasive method using detection of human papillomavirus (HPV) DNA in women's menstrual blood (MB). The participants were divided into 3 cohorts: (i) 235 patients with cervical intraepithelial neoplasia 3 (CIN 3) (n = 48), CIN 2 (n = 60), CIN 1 (n = 58), or condyloma acuminatum (CAC) (n = 69) before treatment or remission; (ii) from the first cohort of patients, 108 CIN 3 or CIN 2 patients after treatment and 62 CIN 1 or CAC patients after remission; and (iii) 323 apparently normal subjects (ANS) without any cervical disease. The HPV genotypes of the infected patients were confirmed by direct sequencing. Quantitative real-time PCR (QRT-PCR) was used to measure the MB HPV16 load for 15 infected patients. Results showed that the sensitivity, specificity, and positive and negative predictive values for detection of MB HPV DNA in samples from patients with CIN or CAC were 82.8%, 93.1%, 90.0%, and 87.9%, respectively. Moreover, MB HPV DNA was found in samples from 22.2% of CIN 3 or CIN 2 patients after treatment, 0.0% of CIN 1 or CAC patients after remission, and 8.1% of ANS, 4 of whom were found to have CIN 1 or CAC. Furthermore, QRT-PCR showed that the normalized MB HPV16 DNA copy numbers in samples from patients with CIN 1 to CIN 3 were significantly increased. These preliminary results suggested that MB HPV DNA is a potential noninvasive marker for these premalignant cervical diseases.

Clinical significance of cytokeratin 20-positive circulating tumor cells detected by a refined immunomagnetic enrichment assay in colorectal cancer patients.

PURPOSE: Current immunomagnetic enrichment method can only detect general epithelial antigens of circulating tumor cells (CTC). Further characterization of the CTCs to provide specific information on the tumor type is not possible. We attempted to overcome this drawback by developing the methodology for using a gastrointestinal-specific anti-cytokeratin (CK) 20 antibody to detect CTCs in colorectal cancer patients' blood. EXPERIMENTAL DESIGN: The protocol was validated using a colorectal cancer SW480 cell line. The clinical significance of findings in colorectal cancer was investigated by detecting CK20-positive CTCs (pCTC) in patients with colorectal cancer, other common cancers, colorectal adenoma, benign colorectal diseases, and normal subjects. Moreover, the malignant nature of CK20 pCTCs was examined by comparing chromosome 17 aberration patterns with those from the corresponding primary tumors. RESULTS: The assay successfully showed CK20-positive SW480 cells. When applied in patient samples, the detection rates were 62% (132 colorectal cancer patients; median number = 11 CTCs), 0% (120 patients with other common cancers), 6% (50 colorectal adenoma patients), 0% (120 patients with benign colorectal diseases), and 0% (40 normal subjects). Furthermore, statistical analysis showed that CK20 pCTC numbers were associated with tumor-node-metastasis stage and lymph node status. Using the median CK20 pCTC numbers as the cutoff points, stratified groups of colorectal cancer patients had significant differences in their recurrence, metastasis, and survival. Finally, chromosome 17 aneusomy in 90% of colorectal cancer patients with CK20 pCTCs matched with those from the primary tumors. CONCLUSIONS: Detection of CK20 pCTCs using the new protocol could generate clinically important information for colorectal cancer patients.

STAT3 activation contributes directly to Epstein-Barr virus-mediated invasiveness of nasopharyngeal cancer cells in vitro.

Nasopharyngeal cancer (NPC) is an Epstein-Barr virus (EBV)-associated head and neck cancer prevalent in Asia. Although with reasons not fully understood, the intrinsic invasiveness of NPC is believed to be EBV-linked. Recently, EBV was found to induce STAT3 activation. Constitutive STAT3 activation correlated with advanced clinical staging in NPC. We hypothesized that STAT3 activation by EBV directly contributes to the intrinsic invasiveness of NPC cells. Phospho-STAT3-Tyr705 was detected in high percentage of NPC tumors (7/10 cases). Using a paired NPC cell line model, HONE-1 and the EBV-infected counterpart, HONE-1-EBV, we found that HONE-1-EBV expressed a higher level of phospho-STAT3-Tyr705 and was approximately 11-fold more invasive than HONE-1. In HONE-1-EBV, STAT3 siRNA targeting inhibited both spontaneous and serum-induced invasion, as well as cell growth. Conversely, activation of STAT3 (by expressing an activated STAT3 mutant, namely STAT3C) in the parental HONE-1, mimicking EBV-induced STAT3 activation, significantly enhanced its invasiveness and proliferation, which was accompanied by increased expression of markers of mesenchymal status, proliferation and anti-apoptosis. Our results demonstrated that EBV-induced STAT3 activation is responsible for NPC cell proliferation and invasion. This was further confirmed by a small molecule inhibitor of JAK/STAT3, JSI-124. JSI-124 inhibited STAT3 activation in HONE-1-EBV, with subsequent growth inhibition, induction of PARP cleavage, abrogation of anchorage-independent growth and invasion. We found that EBV-independent activation of STAT3 by a growth factor, EGF, also contributed to NPC invasion. In conclusion, EBV-induced STAT3 activation directly contributes to the intrinsic invasiveness of NPC cells and STAT3 targeting may be beneficial in treating aggressive NPC.

Advanced proteomic technologies for cancer biomarker discovery.

Proteomic technologies have experienced major improvements in recent years. Such advances have facilitated the discovery of potential tumor markers with improved sensitivities and specificities for the diagnosis, prognosis and treatment monitoring of cancer patients. This review will focus on four state-of-the-art proteomic technologies, namely 2D difference gel electrophoresis, MALDI imaging mass spectrometry, electron transfer dissociation mass spectrometry and reverse-phase protein array. The major advancements these techniques have brought about and examples of their applications in cancer biomarker discovery will be presented in this review, so that readers can appreciate the immense progress in proteomic technologies from 1997 to 2008. Finally, a summary will be presented that discusses current hurdles faced by proteomic researchers, such as the wide dynamic range of protein abundance, standardization of protocols and validation of cancer biomarkers, and a 5-year view of potential solutions to such problems will be provided.

Identification of 5-fluorouracil response proteins in colorectal carcinoma cell line SW480 by two-dimensional electrophoresis and MALDI-TOF mass spectrometry.

Colorectal cancer (CRC) is the second most prevalent cause of cancer-related deaths in the Western world. 5-Fluorouracil (5-FU) is a standard chemotherapeutic drug to treat CRC. However, the response rate is less than 20% and patients who have responded to 5-FU may become resistant. Therefore there is an urgent need to examine the 5-FU response proteins so that patients with no response to 5-FU can change to other treatment strategies promptly. In this study, the proteomic expression profile in a CRC cell line SW480 before and after 5-FU treatment was examined using 2-dimensional electrophoresis technology. Fourteen proteins with differential expression were identified using mass spectrometry and 7 of them were validated using immunocytochemical (ICC) staining. Protein identification indicated that cyclophilin A, cytokeratin 19 (CK19), cytokeratin 8 (CK8), ras-related nuclear protein, heat shock protein 27 (hsp27) and peroxiredoxin 6 (Prx 6) were upregulated whereas heat shock protein 60 (hsp60), cytokeratin 18 (CK18), cytokeratin 9 (CK9), carbamoylphosphate synthetase I, alpha-enolase, heat shock protein 70 (hsp70), nm23 and beta-actin were down-regulated. Seven of the 14 proteins detected were validated by ICC staining, which showed that the expression of hsp27, Prx 6 and hsp70 correlated with that from proteomics profiling. Our results suggest that hsp27, Prx 6 and hsp70 are potential 5-FU response proteins and they may represent potential targets for further evaluation in other 5-FU-sensitive and -resistant CRC cell lines.

Isolated tumor cells and circulating CK20 mRNA in pN0 colorectal cancer patients.

Micrometastases in lymph nodes and blood may provide important prognostic information. In this study, cytokeratin 20 (CK20) positive cells in lymph nodes and circulating CK20 mRNA were studied using 57 paraffin-embedded lymph node specimens and blood from 24 patients with pN0 colorectal cancer (CRC), respectively. Results showed that 29 out of 56 (52%) lymph node specimens had CK20-positive cells (range: 1-35). Follow-up of the patients for 12 months indicated that 4 patients (7%) had CRC metastases to liver, lung, and bone. In addition, 8 out of 24 (33%) samples had at least 2-fold circulating CK20 mRNA expression higher than the pooled normal sample. This study provides evidence that CK20-positive cells were found in the lymph nodes and differentially expressed circulating CK20 mRNA was also detected in the blood from patients with pN0 CRC. Long-term follow-up is necessary to study their prognostic use in patients with non-metastatic CRC.

Clinical studies of immunomodulatory activities of Yunzhi-Danshen in patients with nasopharyngeal carcinoma.

OBJECTIVES: Nasopharyngeal carcinoma (NPC) is a prevalent tumor in Hong Kong. The immune system of such patients could be adversely affected during the course of conventional chemotherapy or radiotherapy. We investigated the immunomodulatory effects of Traditional Chinese Medicine (TCM) Yunzhi-Danshen capsules in NPC patients treated with radiotherapy. DESIGN: Randomized, double-blind, placebo-controlled 16-week study. SETTING/LOCATION: The Prince of Wales Hospital, the Chinese University of Hong Kong, Hong Kong. SUBJECTS: Twenty-seven (27) patients with histologically proven NPC, at least 18 years of age. METHODS: Twenty-seven patients with histologically proven NPC were recruited to take Yunzhi (3.6 g daily) and Dangshem (1.4 g daily) in the form of 12 combination capsules (TCM group) or placebo (12 capsules) daily for 16 weeks, respectively. Flow cytometry was used to assess the percentages and absolute counts of human lymphocyte subsets in whole blood. Plasma concentration of soluble interleukin-2 receptor and soluble tumor necrosis factor receptor 2 was measured by enzyme-linked immunosorbent assay (ELISA). Ex vivo production of tumor necrosis factor-alpha, interleukin (IL)-6, and IL-10 in the whole blood assay culture supernatant was measured by ELISA. RESULTS: The decreases in percentage and absolute count of T lymphocytes in the TCM group were less than those in the placebo group after they took the capsules for 16 weeks (both p < 0.05). Furthermore, the decreases in absolute count of T suppressor cells plus cytotoxic T lymphocytes, and T helper cells in the TCM group were significantly lower than those in the placebo group after they took the capsules for 16 weeks (both p < 0.05). CONCLUSION: These results suggest that Yunzhi-Danshen can exert an immunomodulating effect in alleviating lymphopenia during radiotherapy in NPC patients.

Clinical recommendations for defining platinum unsuitable head and neck cancer patient populations on chemoradiotherapy: A literature review.

Toxicities resulting from platinum based chemotherapy in head and neck cancer is a cause for much concern. There is a lack of clinical criteria for defining these patient populations, which has posed serious problems associated with increased morbidity and consequently an adverse effect on patients' quality of life. In addition, there is a lack of consensus on clinical criteria for defining such patient populations, who may be unsuitable for concurrent chemoradiotherapy. A group of experts in the field of head and neck cancer from the Asia Pacific Region convened in August 2014 in Korea to discuss the development of a set of clinical criteria in order to fill the knowledge gap and provide a reference tool for head and neck oncologists. This paper reports the final output from this meeting and the accompanying literature review, with the aim of aiding clinical decision making with the help of some clinical criteria to identify platinum unsuitable patient populations in head and neck cancer management. Some alternative treatment options are also discussed in this paper.

Jagged 2 silencing inhibits motility and invasiveness of colorectal cancer cell lines.

Although the Notch pathway has been reported to be activated in colorectal cancer (CRC), limited information is available regarding the expression and role of its ligand, Jagged 2 (JAG2), in CRC. Using immunohistochemistry, the present study demonstrated that JAG2 protein expression may be detected in up to 95% of CRC cases and is 3-fold upregulated in tumor cells compared to surrounding normal tissues. This finding suggests that JAG2 may have a role in the tumorigenicity of CRC. To further investigate the cellular functions of JAG2 expression in CRC, two different small interfering RNAs (siRNAs) were used to downregulate JAG2 expression in CRC cell lines (HCT116, DLD-1 and HT-29). The results indicated that JAG2 knockdown inhibits the motility and invasiveness of CRC cell lines without significantly affecting cell proliferation. These findings implicate JAG2 in promoting aggressiveness of CRC, and lay the foundation for its future development as a therapeutic target for the treatment of CRC.

Antitumor effect and enhancement of cytotoxic drug activity by cetuximab in nasopharyngeal carcinoma cells.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is the most common head and neck cancer in southern China and South East Asia. Epidermal growth factor receptor (EGFR) has been proposed as a new target for anticancer therapy. EGFR was over-expressed in 85% of NPC tissues and was associated with poor prognosis. MATERIALS AND METHODS: EGFR protein expression in four NPC cell lines, CNE-2, HONE-1, HK1 and C666-1, was examined by Western immunoblotting. The antitumor effect of cetuximab was studied in the cell lines, either alone or in combination with cisplatin or paclitaxel. RESULTS: EGFR protein expression was highest in the HK1 cell line, moderate in CNE-2 and HONE-1, and lowest in C666-1. Single agent cetuximab demonstrated significant antitumor effect in the HK1 and HONE-1 cell lines, but minimal activity in CNE-2 and C666-1 cells. When cetuximab was combined with cisplatin or paclitaxel in the HK1 and HONE-1 cell lines, an additive enhancement of cytotoxic drug activity was demonstrated. CONCLUSION: Cetuximab demonstrated single agent activity selectively in NPC cell lines with moderate to high EGFR protein expression. Cetuximab could also additively enhance the antitumor effects of cisplatin or paclitaxel in these NPC cell lines. These results support the rationale of combining cetuximab with current standard chemotherapy to further improve the therapeutic ratio in the treatment of NPC. Future studies should aim at defining the predictive markers for response to cetuximab in order to select the responsive tumor for the correctly targeted agents.

Preclinical evaluation of afatinib (BIBW2992) in esophageal squamous cell carcinoma (ESCC).

Esophageal squamous cell carcinoma (ESCC) is the eighth most common cancer worldwide. Epidermal growth factor receptors (EGFR) are often overexpressed in esophageal cancers, thus anti-EGFR inhibitors have been evaluated in ESCC. Afatinib was an irreversible inhibitor of these ErbB family receptors. This study characterized the preclinical activity of afatinib in five ESCC cell lines: HKESC-1, HKESC-2, KYSE510, SLMT-1 and EC-1. ESCC cell lines were sensitive to afatinib with IC50 concentrations at lower micro-molar range (at 72 hour incubation: HKESC-1 = 0.002 µM, HKESC-2 = 0.002 µM, KYSE510 = 1.090 µM, SLMT-1 = 1.161 µM and EC-1 = 0.109 µM) with a maximum growth inhibition over 95%. Afatinib can strongly induce G0/G1 cell cycle arrest in HKESC-2 and EC-1 in a dose- and time-dependent manner. The phosphorylation of ErbB family downstream effectors such as pAKT, pS6 and pMAPK were significantly inhibited in HKESC-2 and EC-1. Apoptosis was observed in both cell lines at 24 hours after exposure to afatinib, as determined by the presence of cleaved PARP. Afatinib could effectively inhibit HKESC-2 tumor growth in mice without obvious toxicity. Afatinib alone has shown excellent growth inhibitory effect on ESCC in both in vitro and in vivo models, however, no synergistic effect was observed when it was combined with chemotherapeutic agents such as 5-fluorouracil (5-FU) and cisplatin. In summary, afatinib can inhibit cell proliferation effectively by arresting the cells in G0/G1 phase, as well as inducing apoptosis in ESCC. These findings warrant further studies of afatinib as therapeutic agent in treating ESCC.

Preclinical evaluation of PI3K inhibitor BYL719 as a single agent and its synergism in combination with cisplatin or MEK inhibitor in nasopharyngeal carcinoma (NPC).

Nasopharyngeal carcinoma (NPC) is endemic to Southeast Asia and over 40% of NPC tissues harbor PIK3CA amplifications. This study aims to study the preclinical activity of a novel PI3K inhibitor, BYL719, in 6 NPC cell lines: C666-1, CNE-2, HK1, HK1-EBV, HONE-1 and HONE-1-LMP1. Over 70% of growth inhibition was attained when NPC cell lines were exposed to increasing concentrations of BYL719, with IC50 values at the low micro-molar range. Two BYL719-sensitive cell lines that harbor PIK3CA mutations, CNE-2 and HONE-1, were selected for further analysis on the effect of BYL719 on cell cycle progression, apoptosis and PI3K signaling. BYL719 significantly reduced the phosphorylation of Akt, and the Akt-mTOR axis downstream effector S6 in these 2 cell lines, but a feedback activation of MAPK was observed at 72 hours post-treatment. BYL719 induced G0/G1 cell cycle arrest and apoptosis in both cell lines. In 3D cell culture models, the growth of NPC spheroids was significantly inhibited in a dose-depending manner. When BYL719 was combined with a MEK inhibitor (AZD6244) in a 3D cell culture system, strong synergism on NPC cell growth was observed with attenuation of MAPK activation. A synergistic inhibitory effect on growth was observed when BYL719 was combined with higher dose levels of cisplatin. These data suggest that BYL719 has preclinical activity in NPC cell lines especially in those which harbor PIK3CA mutation. Combination with a MEK inhibitor maybe a useful strategy that warrants further investigation.

TP53-induced glycolysis and apoptosis regulator promotes proliferation and invasiveness of nasopharyngeal carcinoma cells.

The TP53-induced glycolysis and apoptosis regulator (TIGAR) is the protein product of the p53 target gene, C12orf5. TIGAR blocks glycolysis and promotes cellular metabolism via the pentose phosphate pathway; it promotes the production of cellular nicotinamide adenine dinucleotide phosphate (NADPH), which leads to enhanced scavenging of intracellular reactive oxygen species, and inhibition of oxidative stress-induced apoptosis in normal cells. Our previous study identified a novel nucleoside analog that inhibited cellular growth and induced apoptosis in nasopharyngeal carcinoma (NPC) cell lines via downregulation of TIGAR expression. Furthermore, the growth inhibitory effects of c-Met tyrosine kinase inhibitors were ameliorated by the overexpression of TIGAR in the NPC cell lines. These results indicate a significant role for TIGAR expression in the survival of NPCs. The present study aimed to further define the function of TIGAR expression in NPC cells. In total, 36 formalin-fixed, paraffin-embedded NPC tissue samples were obtained for the immunohistochemical determination of TIGAR expression. The effects of TIGAR expression on cell proliferation, NADPH production and cellular invasiveness were also assessed in NPC cell lines. Overall, TIGAR was overexpressed in 27/36 (75%) of the NPC tissues compared with the adjacent non-cancer epithelial cells. Similarly, TIGAR overexpression was also observed in a panel of six NPC cell lines compared with normal NP460 hTert and Het1A cell lines. TIGAR overexpression led to increased cellular growth, NADPH production and invasiveness of the NPC cell lines, whereas a knockdown of TIGAR expression resulted in significant inhibition of cellular growth and invasiveness. The expression of the two mesenchymal markers, fibronectin and vimentin, was increased by TIGAR overexpression, but reduced following TIGAR-knockdown. The present study revealed that TIGAR overexpression led to increased cellular growth, NADPH production and invasiveness, and the maintenance of a mesenchymal phenotype, in NPC tissues.

Epstein-Barr virus as a paradigm in nasopharyngeal cancer: from lab to clinic.

Nasopharyngeal carcinoma (NPC) of the undifferentiated subtype remains endemic in southern China, with a peak incidence in this region approaching 30 cases per 100,000 population per year. Despite advances in chemotherapy and radiation delivery techniques in localized disease, distant metastasis is still common and NPC remains the seventh leading cause of cancer death in the region. There is great need for early diagnosis, developing novel therapies, and identifying patients with localized disease at higher risk of future recurrence or metastasis to appropriately tailor their treatment and improve outcomes. Knowledge of the integral involvement of Epstein-Barr virus (EBV) in the pathogenesis of undifferentiated NPC has been of seminal importance in developing strategies to optimize disease management. The close association with EBV is being evaluated in multiple settings including screening of at-risk populations, disease prognostication, development of targeted therapies, optimizing adjuvant treatment, and early recurrence detection. These translational studies are likely to have an enormous effect on management of undifferentiated NPC and significantly improve the landscape of the disease in years to come.

Targeting tumor hypoxia in nasopharyngeal carcinoma.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is an endemic head and neck cancer in Southeast Asia. Although concurrent chemoradiotherapy generally results in good clinical response for early diseases, posttreatment relapse and distant metastasis are major causes for NPC deaths. There is an urgent need for more effective therapies for advanced NPC. Recent studies have demonstrated tumor hypoxia can be a therapeutic target in NPC. METHODS: A Medline review of articles published on clinical and preclinical studies targeting tumor hypoxia was performed. RESULTS: Studies published to date showed that tumor hypoxia is common in NPC and it is associated with disease progression and resistance to therapy. Several mechanisms have been proposed on the role of tumor hypoxia in NPC cell survival, angiogenesis, and metabolism. Studies reported by us and by others have demonstrated the therapeutic potential of targeting tumor hypoxia in NPC. CONCLUSIONS: Cumulative studies indicate that targeting tumor hypoxia could be an effective approach for NPC treatment.