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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.

Background Human papillomavirus (HPV) is a known causative factor in the etiology of cervical cancer. Methods HPV DNA genotyping was performed in menstrual blood (MB) collected in napkins from patients with cervical intraepithelial neoplasia (CIN), HPV infection and sexually active apparently normal subjects. In the same patient cohort, MB TAP1 I333V and TAP1 D637G gene polymorphisms were examined. Results The sensitivity, specificity, and positive and negative predictive values of HPV DNA in the detection of CIN or HPV infection were 83% (223 of 268), 98% (131 of 134), 99% (223 of 226), and 74% (131 of 176), respectively. Moreover, HPV DNA was found in 24% (28/118) patients who had loop electrosurgical excision procedure treatment and 0% (0/76) HPV infected or CIN1 patient with proven recovery. On the other hand, the risk of developing high-grade CIN was significantly reduced for AG and GG genotypes compared with AA genotype and for carriers with a G allele compared with those with an A allele for both polymorphisms. Conclusions MB HPV DNA is a potential noninvasive marker for screening and monitoring of squamous intraepithelial lesion. Together with TAP1 I333V and TAP1 D637G gene polymorphisms, the combined test may be useful for stratifying high-risk patients for better follow-up strategies.

The TP53induced 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 stressinduced 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 cMet 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 formalinfixed, paraffinembedded 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 noncancer 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 TIGARknockdown. 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., published_or_final_version

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.

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.

Objectives: Recent evidence has shown that single nucleotide polymorphisms (SNPs) in transporter associated with antigen processing 1 (TAP1) gene includes TAP1 I333V and TAP1 D637G are significantly associated with a protective effect for development of cervical cancer and its pre-malignant states. Hence, TAP1 polymorphisms may potentially be useful to identify women who have less chance to develop high-grade cervical intraepithelial neoplasia (HGCIN) and cervical cancer. Our latest breakthrough in detecting HPV genotypes in the menstrual blood (MB) from patients with CIN and condyloma acuminatum has generated a new era in non-invasive screening for HPV genotypes. Therefore, in this study, we further develop a non-invasive method to predict HGCIN risk in patients with CIN or HPV infection using MB collected in napkin by detecting TAP1 SNP. The information obtained would be important as cervical cancer and CIN are still serious public health problems worldwide especially in developing countries. Materials and Methods: Thirty-eight patients with HGCIN (CIN 2 or 3) and 42 patients with LGCIN (CIN 1) or HPV infection were recruited. MB collected in napkin was put inside ziplock plastic bag which was sent to the laboratory by mail or by hand. Small piece of the napkin was cut out (1 cm x 1 cm x 1 mm) using sterile scissor for DNA extraction. Two SNPs in TAP1 gene (I333V and D637G) were genotyped using polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) technique. The amplified TAP1 gene fragments for I333V and D637G SNP detection were digested by BclI and AccI restriction enzymes, respectively and 3 different genotypes which interpreted as AA (wild type), AG (1 allele showed SNP), and GG (2 alleles showed SNP) were detected at each polymorphic site. Reactions were performed in duplicates and each result was confirmed by DNA sequencing. Results: Both TAP1 polymorphisms in the MB were successfully detected. Their patterns were summarized as a) 3 genotypes at each polymorphic site were detected in our patient cohort (Figure 1, Table 1A); b) the risk to develop HGCIN was significantly reduced for AG and GG genotypes when compared to AA genotype (TAP1 I333V: chi-square test, p = 0.003, odds ratio (OR) = 0.23, 95% confidence interval (CI) = 0.08 to 0.63; TAP1 D637G: chi-square test, p = 0.01, OR = 0.30, 95% CI = 0.12 to 0.76); c) the risk to develop HGCIN was significantly reduced for carriers with a G allele when compared to those with an A allele (Table 1B, TAP1 I333V: chi-square test, p = 0.001, OR = 0.27, 95% CI = 0.12 to 0.62; TAP1 D637G: chi-square test, p = 0.007, OR = 0.36, 95% CI = 0.17 to 0.76). Conclusions: This study is the first to show that MB TAP1 I333V and D637G gene polymorphisms are significantly associated with less risk to develop HGCIN. Acknowledgements: This project was supported by the Direct Grant 2010, The Chinese University of Hong Kong. Citation Format: Sze Chuen Cesar Wong, Thomas Chi Chuen Au, Sammy Chung Sum Chan, Charles Ming Lok Chan, Nancy Bo Yin Tsui, Lawrence Wing Chi Chan, Benjamin Yat Ming Yung. Menstrual blood TAP1 I333V and D637G gene polymorphisms are associated with less risk to develop high-grade cervical intraepithelial neoplasia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1862. doi:10.1158/1538-7445.AM2014-1862

Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus associated cancer of the nasopharynx. Annual incidence in China is high, reaching up to 25 per 100,000. NPC is a highly invasive and metastatic disease. At diagnosis, over 60% of patients have advanced disease where treatment failure due to distant recurrence or metastasis is common. More effective forms of therapy to treat this deadly disease are therefore urgently needed. The TP53 induced glycolysis and apoptosis regulator (TIGAR) is a p53 target gene which blocks glycolysis and promotes cellular metabolism via the pentose phosphate pathway. TIGAR promotes production of cellular nicotinamide adenine dinucleotide phosphate (NADPH), leading to enhanced scavenging of intracellular reactive oxygen species (ROS) and inhibition of oxidative-stress induced apoptosis in normal cells. Previously, we have found that a novel nucleoside analog inhibited cell growth and induced apoptosis in NPC cell lines via down-regulation of TIGAR expression. Moreover, the growth inhibitory effect of c-Met tyrosine kinase inhibitors was ameliorated by over-expression of TIGAR in NPC cell lines. These results are indicative of a pivotal role for TIGAR expression in maintaining NPC cell survival. In this study, we investigated the expression pattern of TIGAR in NPC tumor tissues and the consequences of TIGAR overexpression and knockdown on NPC cell growth and invasion. It was found that TIGAR is overexpressed in 27 out of 36 (75%) NPC cases in the tumor cells compared to their respective normal epithelial cells. Overexpression of TIGAR in NPC cell lines resulted in increased proliferation, invasion, NADPH production and up-regulation of mesenchymal markers including fibronectin and vimentin. These changes were reversed when TIGAR expression was knocked down by transfection with siRNA, demonstrating specificity of the observed effects. Together, our results indicate that TIGAR expression promotes proliferation, invasiveness and expression of mesenchymal markers in NPC cells. These findings may form the basis for development of a new therapeutic target for treatment of NPC in the future. Citation Format: Elaine YL Wong, SC Cesar Wong, Charles ML Chan, Emily KY Lam, Louisa Ho, Cecilia PY Lau, Thomas CC Au, Amanda KC Chan, CM Tsang, George SW Tsao, Vivian WY Lui, Anthony TC Chan. Functional significance of TIGAR expression in nasopharyngeal carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5003. doi:10.1158/1538-7445.AM2014-5003

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.