Sze Chuen Cesar Wong

Expression of frizzled-related protein and Wnt-signalling molecules in invasive human breast tumours

Frizzled‐related protein (Frp) is a new family of secreted proteins that contain a region homologous to the extracellular cysteine‐rich domain (CRD) of the frizzled family proteins. The role of Frp protein is far from clear. To explore the role of Frp and its relationship to the Wnt‐signalling pathway in breast cancer, in situ hybridization and immunohistochemical analyses of Frp, Wnt‐1, APC, β‐catenin, and its target genes c‐myc and cyclin D1 were conducted in 70 specimens of invasive ductal carcinomas of the human breast. Frp mRNA was down‐regulated in 62 and elevated in eight tumour specimens, compared with adjacent normal tissues. In the course of tumour progression, however, Frp mRNA steadily increased in both tumour and the adjacent tissues. Interestingly, the number of cases with axillary lymph node metastasis was significantly lower in the group with elevated Frp than in the group with decreased Frp, suggesting that Frp may contribute as a prognostic factor in invasive breast cancer. Wnt‐1, a gene implicated in human breast cancer, was markedly elevated in grade 1 tumours, but declined as tumour grade declined. The level of Wnt‐1 was linearly correlated with its downstream target β‐catenin (p<0.05), but was inversely correlated with Frp (p<0.05), suggesting a possible negative regulatory role of Frp with regard to Wnt‐1. APC was inversely correlated with β‐catenin (p<0.05). β‐catenin, a key transcriptional activator responsible for the activation of both c‐myc and cyclin D1 in colorectal tumours, was detected at high levels in the plasma membranes of cells in normal tissue. In tumour masses, however, β‐catenin lost its tight association with the membrane and diffused into the cytoplasm. Surprisingly, it clearly did not penetrate the nuclei, despite the fact that both c‐myc and cyclin D1 were markedly elevated in all tumour tissues. As revealed in this study, Wnt‐1/β‐catenin plays very different roles in the oncogenesis of breast and colon cancers. This first systemic analysis of the Frp and the Wnt‐signalling pathway in human breast cancer provides a springboard for further work on the role of Frp in the development of breast cancer. Copyright

WNT5A Exhibits Tumor-Suppressive Activity through Antagonizing the Wnt/β-Catenin Signaling, and Is Frequently Methylated in Colorectal Cancer

Purpose: Aberrant activation of the Wnt/β-catenin signaling pathway is associated with multiple tumors including colorectal cancer (CRC). WNT5A is a member of the nontransforming Wnt protein family, whose role in tumorigenesis is still ambiguous. We investigated its epigenetic alteration in CRCs. Experimental Design: We examined its expression and methylation in normal colon, CRC cell lines, and tumors. We also evaluated its tumor-suppressive function and its modulation to Wnt signaling in CRC cells. Results:WNT5A is silenced in most CRC cell lines due to promoter methylation, but is expressed in most normal tissues including the colon, and is unmethylated in normal colon epithelial cells. WNT5A expression could be reactivated by pharmacologic or genetic demethylation, indicating that methylation directly mediates its silencing. WNT5A methylation was frequently detected in CRC tumors (14 of 29, 48%), but only occasionally in paired normal colon tissues (2 of 15, 13%; P = 0.025). Ectopic expression of WNT5A, but not its nonfunctional short-isoform with the WNT domain deleted, in silenced CRC cells resulted in substantial inhibition of tumor cell clonogenicity, which is associated with down-regulated intracellular β-catenin protein level and concomitant decrease in β-catenin activity. Conclusions:WNT5A is frequently inactivated in CRC by tumor-specific methylation, and thus, is a potential biomarker. WNT5A could act as a tumor suppressor for CRC by antagonizing the Wnt/β-catenin signaling.

Prognostic and Diagnostic Significance of β-Catenin Nuclear Immunostaining in Colorectal Cancer

In the present study, we investigated the prognostic and diagnostic significance of β-catenin nuclear immunostaining in 60 specimens of normal colorectal tissue; 180 specimens of colorectal polyps, adenomas, and carcinomas; and 40 specimens from patients with the simultaneous occurrence of polyps, adenomas, and carcinomas. Additional specimens from 59 patients with colorectal carcinoma and 14 patients with adenoma who subsequently developed carcinoma were examined for possible survival study. Immunohistochemical staining showed that the occurrence of nuclear β-catenin correlated with the sequential stages in colorectal carcinogenesis, in which positive staining was observed in 0% of normal tissues, 8% of polyps, 92% of adenomas, and 100% of carcinomas. High immunohistochemical scores in colorectal carcinoma were significantly associated with lymph node metastasis and poor survival. Adenomas associated with synchronous or metachronous carcinomas showed significantly higher levels of nuclear β-catenin compared with adenomas without associated carcinomas. Nuclear translocation of β-catenin was rare or absent in other types of cytokeratin 20 positive adenocarcinomas examined (99 cases). Thus, it was positive in only 7% of colonic mucinous adenocarcinomas, 3% of pancreatic adenocarcinomas, 8% of ovarian mucinous cystadenocarcinomas, and 0% of gastric adenocarcinomas. However, 100% of primary and metastatic colorectal adenocarcinomas were positive for nuclear staining for β-catenin. Thus, nuclear staining for β-catenin may serve as an additional parameter to help distinguish colorectal adenocarcinomas from adenocarcinomas of other tissue sites. Collectively, the present large-scale study has clearly addressed the clinical significance of β-catenin nuclear translocation with respect to tumor progression, survival, and differential diagnosis.

Quantification of Plasma β-Catenin mRNA in Colorectal Cancer and Adenoma Patients

Purpose: Colorectal cancer is an important cause of cancer deaths. Here, we focused our investigation on the β-catenin gene which is implicated in colorectal carcinogenesis and tested whether β-catenin mRNA is detectable in the plasma of colorectal carcinoma and adenoma patients using quantitative reverse transcriptase-PCR. Experimental Design: Plasma β-catenin mRNA was measured from 58 colorectal carcinoma patients, 49 colorectal adenoma patients, and 43 apparently normal subjects using intron-spanning primers and Taqman probes. Five clinicopathological parameters were studied and correlated with plasma β-catenin mRNA concentration. Additionally, 19 colorectal carcinoma patients after tumor removal were also recruited for plasma β-catenin mRNA measurement to further demonstrate the clinical usefulness of this test. Results: β-catenin mRNA was detected with median concentrations of 8737 (range: 1480–933,100), 1218 (range: 541–2,254) and 291 (range: 0–1,366) copies/ml plasma in colorectal carcinoma, colorectal adenoma, and apparently normal subjects, respectively. Statistical analysis demonstrated that plasma β-catenin mRNA concentration was correlated to tumor stage but not sex, age, lymph node status, and degree in differentiation. Moreover, plasma β-catenin mRNA concentration decreased significantly after tumor removal in 16 of 19 (84%) colorectal carcinoma patients. Conclusions: We conclude that plasma β-catenin mRNA may potentially serve as a marker for colorectal cancer.

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.

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 ∼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.

Disease-Specific Target Gene Expression Profiling of Molecular Imaging Probes: Database Development and Clinical Validation

Molecular imaging probes can target abnormal gene expression patterns in patients and allow early diagnosis of disease. For selecting a suitable imaging probe, the current Molecular Imaging and Contrast Agent Database (MICAD) provides descriptive and qualitative information on imaging probe characteristics and properties. However, MICAD does not support linkage with the expression profiles of target genes. The proposed Disease-specific Imaging Probe Profiling (DIPP) database quantitatively archives and presents the gene expression profiles of targets across different diseases, anatomic regions, and subcellular locations, providing an objective reference for selecting imaging probes. The DIPP database was validated with a clinical positron emission tomography (PET) study on lung cancer and an in vitro study on neuroendocrine cancer. The retrieved records show that choline kinase beta and glucose transporters were positively and significantly associated with lung cancer among the targets of 11C-choline and [18F]fluoro-2-deoxy-2-d-glucose (FDG), respectively. Their significant overexpressions corresponded to the findings that the uptake rate of FDG increased with tumor size but that of 11C-choline remained constant. Validated with the in vitro study, the expression profiles of disease-associated targets can indicate the eligibility of patients for clinical trials of the treatment probe. A Web search tool of the DIPP database is available at http://www.polyu.edu.hk/bmi/dipp/.Molecular imaging probes can target abnormal gene expression patterns in patients and allow early diagnosis of disease. For selecting a suitable imaging probe, the current Molecular Imaging and Contrast Agent Database (MICAD) provides descriptive and qualitative information on imaging probe characteristics and properties. However, MICAD does not support linkage with the expression profiles of target genes. The proposed Disease-specific Imaging Probe Profiling (DIPP) database quantitatively archives and presents the gene expression profiles of targets across different diseases, anatomic regions, and subcellular locations, providing an objective reference for selecting imaging probes. The DIPP database was validated with a clinical positron emission tomography (PET) study on lung cancer and an in vitro study on neuroendocrine cancer. The retrieved records show that choline kinase beta and glucose transporters were positively and significantly associated with lung cancer among the targets of 11C-choline and [18F]fluoro-2- deoxy-2-D-glucose (FDG), respectively. Their significant overexpressions corresponded to the findings that the uptake rate of FDG increased with tumor size but that of 11C-choline remained constant. Validated with the in vitro study, the expression profiles of disease-associated targets can indicate the eligibility of patients for clinical trials of the treatment probe. A Web search tool of the DIPP database is available at http://www.polyu.edu.hk/bmi/dipp/.

The contribution of bifunctional skipDewax pretreatment solution, rabbit monoclonal antibodies, and polymer detection systems in immunohistochemistry

CONTEXT In immunohistochemistry, nonstandardized antigen retrieval protocols and fluids, poor-quality antibodies, and the presence of endogenous biotin frequently lead to incorrect results. Recently, advanced reagents including bifunctional SkipDewax pretreatment solution (BSPS), rabbit monoclonal (RM) antibodies, and biotin-free polymer detection systems (PDSs) have been developed, which, it is claimed, resolve these problems. OBJECTIVES To determine whether BSPS, RM antibodies, and biotin-free PDSs improve the accuracy of immunohistochemistry; to optimize a new protocol consisting of a combination of BSPS, RM antibodies, and PDSs; and to compare it with a conventional protocol. DESIGN The efficacies of BSPS, RM antibodies, and PDSs were compared with those of their respective conventional reagents using multitissue spring-roll sections. The new protocol was compared with a conventional protocol using Ki-67 immunostaining of 49 colorectal carcinoma specimens. RESULTS For antigen retrieval, BSPS resulted in similar or better tissue staining than an EDTA solution, but the efficacy of BSPS decreased when it was reused. Most RM antibodies resulted in a greater proportion of positive cells than the corresponding non-RM antibodies, which did not produce satisfactory results in the absence of antigen retrieval. The PDSs Bond, ChemMate, and SuperPicture resulted in a high percentage of positive cells, good staining intensities, and low backgrounds. Other PDSs, except that from Ventana, resulted in high backgrounds and false positivity. The new combined protocol resulted in better Ki-67 staining than the conventional assay. CONCLUSIONS Bifunctional SkipDewax pretreatment solution, RM antibodies, and PDSs improve staining quality and diagnostic accuracy of immunohistochemistry assays and provide a foundation for standardization.

An optimised protocol for the extraction of non-viral mRNA from human plasma frozen for three years

Aims: To detect non-viral mRNA in human plasma that has been frozen for three years using a new protocol. Methods: Plasma from 15 patients with colorectal cancer and 10 normal subjects was separated and frozen with Trizol at −80°C for three years. As a control measure, plasma from 10 of the 15 patients was separated using the same protocol but no Trizol during storage. After three years, all samples were extracted using Trizol and RNeasy before the reverse transcriptase polymerase chain reaction was performed to detect non-viral β catenin mRNA. In addition, extraction of three plasma samples by Trizol or RNeasy independently was carried out for comparison. Results: β Catenin mRNA was detected in all 15 patient plasma samples and only one of the 10 normal subjects. In contrast, no β catenin mRNA was found in the control and patient samples that were independently extracted by Trizol and RNeasy kit. Conclusions: This new protocol is a reliable method for extracting non-viral mRNA from the plasma of patients with cancer after longterm storage for three years. Extractions using Trizol and RNeasy kits independently could not isolate mRNA with sufficient quantity and quality for detection.

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

SummaryRecent 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.