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We have developed an optimized protocol for plasma targeted mRNA sequencing in our previous study. Here, we performed plasma targeted mRNA sequencing for 40 colorectal adenoma patients and 39 colonoscopy-proven normal controls in order to find potential circulating mRNA markers for colorectal adenoma. Results showed that GSK3A and RHOA were differential expressed genes identified by a cut-off of fold change >2 and adjusted P value GSK3A (0.01-fold with adjusted P −6) and RHOA (0.35-fold with adjusted P

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.

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.

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.

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.

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.

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