Publications by authors named "Ming Hui Lee"

38 Publications

Risk Factors of Thyroid Eye Disease.

Endocr Pract 2020 Dec 14. Epub 2020 Dec 14.

Yong Loo Lin School of Medicine, National University of Singapore, Singapore; Department of Medicine, National University Hospital, Singapore. Electronic address:

Objective: To examine risk factors that might be associated with thyroid eye disease (TED) in patients with Graves' disease (GD), which may guide physicians in the prevention and management of TED.

Methods: Medline and Embase were searched for articles discussing risk factors of TED. Comparisons were made between GD patients with and without TED, and between active and inactive TED GD patients. Weighted mean differences (WMDs) and odds ratios (ORs) were determined for continuous and dichotomous outcomes, respectively. Results were pooled with random effects using the DerSimonian and Laird model.

Results: Fifty-six articles were included in the analysis. Smoking, inclusive of current and previous smoking status, was a significant risk factor for TED (OR: 2.401; CI: 1.958-2.945; P < .001). Statistical significance was found upon meta-regression between male sex and the odds of smoking and TED (β = 1.195; SE = 0.436; P = .013). Other risk factors were also examined, and patients with TED were significantly older than those without TED (WMD: 1.350; CI: 0.328-2.372; P = .010). While both age (WMD: 5.546; CI: 3.075-8.017; P < .001) and male sex (OR: 1.819; CI: 1.178-2.808; P = .007) were found to be significant risk factors for active TED patients compared to inactive TED patients, no statistical significance was found for family history, thyroid status, cholesterol levels, or body mass index.

Conclusion: Factors such as smoking, sex, and age predispose GD patients to TED, and TED patients to active TED. A targeted approach in the management of GD and TED is required to reduce the modifiable risk factor of smoking.
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http://dx.doi.org/10.1016/j.eprac.2020.11.011DOI Listing
December 2020

"3G" Trial: An RNA Editing Signature to Guide Gastric Cancer Chemotherapy.

Cancer Res 2021 Feb 8. Epub 2021 Feb 8.

Singapore Gastric Cancer Consortium.

Gastric cancer (GC) cases are often diagnosed at an advanced stage with poor prognosis. Platinum-based chemotherapy has been internationally accepted as first-line therapy for inoperable or metastatic GC. To achieve greater benefits, selection of patients eligible for this treatment is critical. Although gene expression profiling has been widely used as a genomic classifier to identify molecular subtypes of GC and to stratify patients for different chemotherapy regimens, its prediction accuracy can be improved. Adenosine-to-inosine (A-to-I) RNA editing has emerged as a new player contributing to GC development and progression, offering potential clinical utility for diagnosis and treatment. Using a systematic computational approach followed by both in vitro validations and in silico validations in TCGA, we conducted a transcriptome-wide RNA editing analysis of a cohort of 104 patients with advanced GC and identified an RNA editing (GCRE) signature to guide GC chemotherapy. RNA editing events stood as a prognostic and predictive biomarker in advanced GC. A GCRE score based on the GCRE signature consisted of 50 editing sites associated with 29 genes, predicting response to chemotherapy with a high accuracy (84%). Of note, patients demonstrating higher editing levels of this panel of sites presented a better overall response. Consistently, GC cell lines with higher editing levels showed higher chemosensitivity. Applying the GCRE score on TCGA dataset confirmed that responders had significantly higher levels of editing in advanced GC. Overall, this newly defined GCRE signature reliably stratifies patients with advanced GC and predicts response from chemotherapy.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-2872DOI Listing
February 2021

A Polypeptide of Tumor-Associated Antigen L6 with Intrinsic Adjuvant Activity Enhances Antitumor Immunity.

Vaccines (Basel) 2020 Oct 21;8(4). Epub 2020 Oct 21.

Graduate Institute of Biomedical Sciences, China Medical University, Taichung 404, Taiwan.

Peptide vaccines are safe, and aim to elicit and expand tumor-specific immunity so as to eradicate tumors. However, achieving strong and long-lasting anti-tumor immunity with peptide vaccines for the antigen-specific treatment of cancer is challenging, in part because their efficacy depends on strong adjuvants or immunomodulators. We approached this problem by conjugating an epitope-based cancer vaccine with a lipidated sequence (an immunomodulator) to elicit a strong immune response. Lipidated and non-lipidated polyepitope proteins were generated that contained the universal T helper cell epitope (pan-DR), B cell epitopes, and the extended loop sequence of extracellular domain 2 of tumor-associated antigen L6 (TAL6). We show that the lipidated polyepitope cancer vaccine can activate bone marrow-derived dendritic cells, and trigger effective antigen-specific antibody and T helper cell responses, more effectively than the non-lipidated vaccine. Moreover, potent T cell immune responses were elicited in mice inoculated with the lipidated polyepitope cancer vaccine, providing protective antitumor immunity in mice bearing TAL6 tumors. Our study demonstrates that a lipidated polyepitope cancer vaccine could be employed to generate potent anti-tumor immune responses, including humoral and cellular immunity, which could be beneficial in the treatment of TAL6 cancer.
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http://dx.doi.org/10.3390/vaccines8040620DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7711899PMC
October 2020

Therapeutic Zfra4-10 or WWOX7-21 Peptide Induces Complex Formation of WWOX with Selective Protein Targets in Organs that Leads to Cancer Suppression and Spleen Cytotoxic Memory Z Cell Activation In Vivo.

Cancers (Basel) 2020 Aug 5;12(8). Epub 2020 Aug 5.

Laboratory of Molecular Immunology, Institute of Molecular Medicine, National Cheng Kung University, College of Medicine, Tainan 70101 Taiwan.

Synthetic Zfra4-10 and WWOX7-21 peptides strongly suppress cancer growth in vivo. Hypothetically, Zfra4-10 binds to the membrane Hyal-2 of spleen Z cells and activates the Hyal-2/WWOX/SMAD4 signaling for cytotoxic Z cell activation to kill cancer cells. Stimulation of membrane WWOX in the signaling complex by a WWOX epitope peptide, WWOX7-21, is likely to activate the signaling. Here, mice receiving Zfra4-10 or WWOX7-21 peptide alone exhibited an increased binding of endogenous tumor suppressor WWOX with ERK, C1qBP, NF-κB, Iba1, p21, CD133, JNK1, COX2, Oct4, and GFAP in the spleen, brain, and/or lung which led to cancer suppression. However, when in combination, Zfra4-10 and WWOX7-21 reduced the binding of WWOX with target proteins and allowed tumor growth in vivo. In addition to Zfra4-10 and WWOX7-21 peptides, stimulating the membrane Hyal-2/WWOX complex with Hyal-2 antibody and sonicated hyaluronan (HAson) induced Z cell activation for killing cancer cells in vivo and in vitro. Mechanistically, Zfra4-10 binds to membrane Hyal-2, induces dephosphorylation of WWOX at pY33 and pY61, and drives Z cell activation for the anticancer response. Thus, Zfra4-10 and WWOX7-21 peptides, HAson, and the Hyal-2 antibody are of therapeutic potential for cancer suppression.
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http://dx.doi.org/10.3390/cancers12082189DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7464583PMC
August 2020

Prevalence of thyroid eye disease in Graves' disease: A meta-analysis and systematic review.

Clin Endocrinol (Oxf) 2020 Oct 10;93(4):363-374. Epub 2020 Aug 10.

Yong Loo Lin School of Medicine, National University of Singapore, Singapore.

Objective: Thyroid eye disease (TED) is a debilitating condition that frequently manifests in patients suffering from Graves' disease (GD). This study aims to analyse the prevalence of TED among GD patients, with a focus on geographical region-specific rates.

Methods: Medline and Embase were searched for articles examining TED prevalence on April 2020, and articles were retrieved and sieved. Statistical analysis was performed after Freeman-Tukey double arcsine transformation. Thereafter, results were pooled with random effects by DerSimonian and Laird model.

Results: Fifty-seven articles involving 26,804 patients were included in the review. The overall pooled prevalence of TED was 40% (CI: 0.32 to 0.48) and by continent was 38% (CI: 0.31 to 0.46) for Europe, 44% (CI: 0.32 to 0.56) for Asia, 27% (CI: 0.06 to 0.56) for North America and 58% (CI: 0.55 to 0.61) for Oceania. The prevalence of TED in Southeast Asia was 35% (CI: 0.24 to 0.47) and Middle East 48% (CI: 0.19 to 0.78). Subgroup analysis showed regions with predominantly Caucasians (37%; CI: 0.28 to 0.46) had a lower prevalence of TED compared to Asians (45%; CI: 0.33 to 0.58). The pooled prevalence of lid retraction was 57% (CI: 0.39 to 0.74), proptosis 57% (CI: 0.48 to 0.65), diplopia 36% (CI: 0.24 to 0.48) and ocular hypertension 13% (CI: 0.06 to 0.19).

Conclusion: A substantial proportion of patients with GD have TED and often manifest as lid retraction, proptosis and diplopia. Early detection through active screening might help to mitigate the progression of TED and its associated complications.
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http://dx.doi.org/10.1111/cen.14296DOI Listing
October 2020

Genomic and epigenomic EBF1 alterations modulate TERT expression in gastric cancer.

J Clin Invest 2020 06;130(6):3005-3020

Cancer and Stem Cell Biology Program, Duke-NUS Medical School, Singapore.

Transcriptional reactivation of telomerase catalytic subunit (TERT) is a frequent hallmark of cancer, occurring in 90% of human malignancies. However, specific mechanisms driving TERT reactivation remain obscure for many tumor types and in particular gastric cancer (GC), a leading cause of global cancer mortality. Here, through comprehensive genomic and epigenomic analysis of primary GCs and GC cell lines, we identified the transcription factor early B cell factor 1 (EBF1) as a TERT transcriptional repressor and inactivation of EBF1 function as a major cause of TERT upregulation. Abolishment of EBF1 function occurs through 3 distinct (epi)genomic mechanisms. First, EBF1 is epigenetically silenced via DNA methyltransferase, polycomb-repressive complex 2 (PRC2), and histone deacetylase activity in GCs. Second, recurrent, somatic, and heterozygous EBF1 DNA-binding domain mutations result in the production of dominant-negative EBF1 isoforms. Third, more rarely, genomic deletions and rearrangements proximal to the TERT promoter remobilize or abolish EBF1-binding sites, derepressing TERT and leading to high TERT expression. EBF1 is also functionally required for various malignant phenotypes in vitro and in vivo, highlighting its importance for GC development. These results indicate that multimodal genomic and epigenomic alterations underpin TERT reactivation in GC, converging on transcriptional repressors such as EBF1.
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http://dx.doi.org/10.1172/JCI126726DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7260007PMC
June 2020

Liposomal TLR9 Agonist Combined with TLR2 Agonist-Fused Antigen Can Modulate Tumor Microenvironment through Dendritic Cells.

Cancers (Basel) 2020 Mar 28;12(4). Epub 2020 Mar 28.

National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Miaoli 350401, Taiwan.

Dendritic cells (DCs) are antigen-presenting cells involved in T cell activation and differentiation to regulate immune responses. Lipoimmunogens can be developed as pharmaceutical lipoproteins for cancer immunotherapy to target DCs via toll-like receptor 2 (TLR2) signaling. Previously, we constructed a lipoimmunogen, a lipidated human papillomavirus (HPV) E7 inactive mutant (rlipoE7m), to inhibit the growth of HPV16 E7-expressing tumor cells in a murine model. Moreover, this antitumor effect could be enhanced by a combinatory treatment with CpG oligodeoxynucleotides (ODN). To improve safety, we developed a rlipoE7m plus DOTAP liposome-encapsulated native phosphodiester CpG (POCpG/DOTAP) treatment to target DCs to enhance antitumor immunity. We optimized the formulation of rlipoE7m and POCpG/DOTAP liposomes to promote conventional DC and plasmacytoid DC maturation in vitro and in vivo. Combination of rlipoE7m plus POCpG/DOTAP could activate conventional DCs and plasmacytoid DCs to augment IL-12 production to promote antitumor responses by intravenous injection. In addition, the combination of rlipoE7m plus POCpG/DOTAP could elicit robust cytotoxic T lymphocytes (CTLs) by intravenous immunization. Interestingly, the combination of rlipoE7m plus POCpG/DOTAP could efficiently inhibit tumor growth via intravenous immunization. Moreover, rlipoE7m plus POCpG/DOTAP combined reduced the number of tumor-infiltrating regulatory T cells dramatically due to downregulation of IL-10 production by DCs. These results showed that the combination of rlipoE7m plus POCpG/DOTAP could target DCs via intravenous delivery to enhance antitumor immunity and reduce the number of immunosuppressive cells in the tumor microenvironment.
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http://dx.doi.org/10.3390/cancers12040810DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7225995PMC
March 2020

WWOX Possesses -Terminal Cell Surface-Exposed Epitopes WWOX and WWOX for Signaling Cancer Growth Suppression and Prevention In Vivo.

Cancers (Basel) 2019 Nov 19;11(11). Epub 2019 Nov 19.

Laboratory of Molecular Immunology, Institute of Molecular Medicine, College of Medicine, National Cheng Kung University, Tainan 70101, Taiwan.

Membrane hyaluronidase Hyal-2 supports cancer cell growth. Inhibition of Hyal-2 by specific antibody against Hyal-2 or pY216-Hyal-2 leads to cancer growth suppression and prevention in vivo. By immunoelectron microscopy, tumor suppressor WWOX is shown to be anchored, in part, in the cell membrane by Hyal-2. Alternatively, WWOX undergoes self-polymerization and localizes in the cell membrane. Proapoptotic pY33-WWOX binds Hyal-2, and TGF-β induces internalization of the pY33-WWOX/Hyal-2 complex to the nucleus for causing cell death. In contrast, when pY33 is downregulated and pS14 upregulated in WWOX, pS14-WWOX supports cancer growth in vivo. Here, we investigated whether membrane WWOX receives extracellular signals via surface-exposed epitopes, especially at the S14 area, that signals for cancer growth suppression and prevention. By using a simulated 3-dimentional structure and generated specific antibodies, WWOX epitopes were determined at amino acid #7 to 21 and #286 to 299. Synthetic WWOX7-21 peptide, or truncation to 5-amino acid WWOX7-11, significantly suppressed and prevented the growth and metastasis of melanoma and skin cancer cells in mice. Time-lapse microscopy revealed that WWOX7-21 peptide potently enhanced the explosion and death of 4T1 breast cancer stem cell spheres by ceritinib. This is due to rapid upregulation of proapoptotic pY33-WWOX, downregulation of prosurvival pERK, prompt increases in Ca influx, and disruption of the IkBα/WWOX/ERK prosurvival signaling. In contrast, pS14-WWOX7-21 peptide dramatically increased cancer growth in vivo and protected cancer cells from ceritinib-mediated apoptosis in vitro, due to a prolonged ERK phosphorylation. Further, specific antibody against pS14-WWOX significantly enhanced the ceritinib-induced apoptosis. Together, the -terminal epitopes WWOX7-21 and WWOX7-11 are potent in blocking cancer growth in vivo. WWOX7-21 and WWOX7-11 peptides and pS14-WWOX antibody are of therapeutic values in suppressing and preventing cancer growth in vivo.
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http://dx.doi.org/10.3390/cancers11111818DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6895976PMC
November 2019

DNA epigenetic signature predictive of benefit from neoadjuvant chemotherapy in oesophageal adenocarcinoma: results from the MRC OE02 trial.

Eur J Cancer 2019 12 23;123:48-57. Epub 2019 Oct 23.

Cancer and Stem Cell Biology Program, Duke-NUS Medical School, Singapore; Cancer Science Institute of Singapore, National University of Singapore, Singapore; Biomedical Research Council, Agency for Science, Technology and Research, Singapore; SingHealth/Duke-NUS Institute of Precision Medicine, National Heart Centre Singapore, Singapore. Electronic address:

Background: DNA methylation signatures describing distinct histological subtypes of oesophageal cancer have been reported. We studied DNA methylation in samples from the MRC OE02 phase III trial, which randomised patients with resectable oesophageal cancer to surgery alone (S) or neoadjuvant chemotherapy followed by surgery (CS).

Aim: The aim of the study was to identify epigenetic signatures predictive of chemotherapy benefit in patients with oesophageal adenocarcinoma (OAC) from the OE02 trial and validate the findings in an independent cohort.

Methods: DNA methylation was analysed using the Illumina GoldenGate platform on surgically resected OAC specimens from patients in the OE02 trial. Cox proportional hazard analysis was performed to select probes predictive of survival in the CS arm. Non-negative matrix factorisation was used to perform clustering and delineate DNA methylation signatures. The findings were validated in an independent cohort of patients with gastroesophageal adenocarcinoma treated with neoadjuvant chemotherapy.

Results: A total of 229 patients with OAC were analysed from the OE02 trial (118 in the CS arm and 111 in the S arm). There was no difference in DNA methylation status between the CS and S arms. A metagene signature was created by dichotomising samples into two clusters. In cluster 1, patients in the CS arm had significant overall survival (OS) benefit (median OS CS: 931 days vs. S: 536 days [HR: 1.54, P = 0.031]). In cluster 2, patients in the CS arm had similar (or worse) OS compared with patients in the S arm (CS: 348 days vs. S: 472 days [HR: 0.70, P = 0.1], and test of interaction was significant (p = 0.005). In the validation cohort (n = 13), there was no difference in DNA methylation status in paired pre- and post-treatment samples. When the epigenetic signature was applied, cluster 1 samples had better OS (median OS, cluster 1: 1174 days vs. cluster 2: 392 days, HR: 3.47, p = 0.059) CONCLUSIONS: This is the first and largest study of DNA methylation in patients with OAC uniformly treated in a randomised phase III trial. We identified an epigenetic signature that may serve as a predictive biomarker for chemotherapy benefit in OAC.
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http://dx.doi.org/10.1016/j.ejca.2019.09.016DOI Listing
December 2019

A p53/TIAF1/WWOX triad exerts cancer suppression but may cause brain protein aggregation due to p53/WWOX functional antagonism.

Cell Commun Signal 2019 07 17;17(1):76. Epub 2019 Jul 17.

Institute of Molecular Medicine, National Cheng Kung University, College of Medicine, Tainan, Taiwan, 70101, Republic of China.

Background: Tumor suppressor WWOX physically binds p53 and TIAF1 and together induces apoptosis and tumor suppression. To understand the molecular action, here we investigated the formation of WWOX/TIAF1/p53 triad and its regulation of cancer cell migration, anchorage-independent growth, SMAD promoter activation, apoptosis, and potential role in neurodegeneration.

Methods: Time-lapse microscopy was used to measure the extent of cell migration. Protein/protein interactions were determined by co-immunoprecipitation, FRET microscopy, and yeast two-hybrid analysis. The WWOX/TIAF1/p53 triad-mediated cancer suppression was determined by measuring the extent of cell migration, anchorage-independent growth, SMAD promoter activation, and apoptosis. p53-deficient lung cancer cell growth in nude mice was carried out to assess the tumor suppressor function of ectopic p53 and/or WWOX.

Results: Wwox-deficient MEF cells exhibited constitutive Smad3 and p38 activation and migrated individually and much faster than wild type cells. TGF-β increased the migration of wild type MEF cells, but significantly suppressed Wwox knockout cell migration. While each of the triad proteins is responsive to TGF-β stimulation, ectopically expressed triad proteins suppressed cancer cell migration, anchorage-independent growth, and SMAD promoter activation, as well as caused apoptosis. The effects are due in part to TIAF1 polymerization and its retention of p53 and WWOX in the cytoplasm. p53 and TIAF1 were effective in suppressing anchorage-independent growth, and WWOX ineffective. p53 and TIAF1 blocked WWOX or Smad4-regulated SMAD promoter activation. WWOX suppressed lung cancer NCI-H1299 growth and inhibited splenomegaly by inflammatory immune response, and p53 blocked the event in nude mice. The p53/WWOX-cancer mice exhibited BACE upregulation, APP degradation, tau tangle formation, and amyloid β generation in the brain and lung.

Conclusion: The WWOX/TIAF1/p53 triad is potent in cancer suppression by blocking cancer cell migration, anchorage-independent growth and SMAD promoter activation, and causing apoptosis. Yet, p53 may functionally antagonize with WWOX. p53 blocks WWOX inhibition of inflammatory immune response induced by cancer, and this leads to protein aggregation in the brain as seen in the Alzheimer's disease and other neurodegeneration.
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http://dx.doi.org/10.1186/s12964-019-0382-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6637503PMC
July 2019

pathway mapping identifies wild-type as a targetable metabolic node in gastric cancer.

Gut 2020 02 8;69(2):231-242. Epub 2019 May 8.

Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore.

Objective: Gastric cancer (GC) is a leading cause of cancer mortality. Previous studies have shown that is specifically overexpressed in GC and functionally required for GC development. In this study, we investigated, on a genome-wide scale, target genes of HNF4α and oncogenic pathways driven by HNF4α and HNF4α target genes.

Design: We performed HNF4α chromatin immunoprecipitation followed by sequencing across multiple GC cell lines, integrating HNF4α occupancy data with (epi)genomic and transcriptome data of primary GCs to define HNF4α target genes of and relevance. To investigate mechanistic roles of and HNF4α targets, we performed cancer metabolic measurements, drug treatments and functional assays including murine xenograft experiments.

Results: Gene expression analysis across 19 tumour types revealed to be specifically upregulated in GCs. Unbiased pathway analysis revealed organic acid metabolism as the top -regulated pathway, orthogonally supported by metabolomic analysis. Isocitrate dehydrogenase 1 () emerged as a convergent direct target gene regulating GC metabolism. We show that wild-type is essential for GC cell survival, and that certain GC cells can be targeted by IDH1 inhibitors.

Conclusions: Our results highlight a role for in sustaining GC oncogenic metabolism, through the regulation of . Drugs targeting wild-type may thus have clinical utility in GCs exhibiting overexpression, expanding the role of in cancer beyond mutated malignancies.
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http://dx.doi.org/10.1136/gutjnl-2018-318025DOI Listing
February 2020

A therapeutic vaccine targeting HPV E6/E7 with intrinsic Toll-like receptor 2 agonist activity induces antitumor immunity.

Am J Cancer Res 2018 1;8(12):2528-2537. Epub 2018 Dec 1.

Graduate Institute of Biomedical Sciences, China Medical University Taichung, Taiwan.

The E6 and E7 oncoproteins of human papillomavirus (HPV) are ideal targets for developing immunotherapeutic approaches to treat HPV-associated tumors. Our previous studies showed that a recombinant lipidated HPV16 E7 mutant (rlipo-E7m) with inactivation of the E7 oncogenic functions can activate antigen presenting cells through Toll-like receptor 2 (TLR2) and induce antitumor immunity. Given that some HPV-associated tumors overexpress E6 but not E7, it is necessary to include therapeutic agents containing HPV E6 in therapeutic vaccine development to broaden the utility of the vaccine. In this study, we further incorporated a mutant HPV16 E6 (E6m) into rlipo-E7m to generate rlipo-E6mE7m, which could elicit both E6- and E7-specific immune responses after immunization. The rlipo-E6mE7m immunization induced higher levels of T cell proliferation and cytotoxic T lymphocyte response than the nonlipidated recombinant E6mE7m (rE6mE7m) immunization. Accordingly, a single-dose administration of rlipo-E6mE7m at day 7 after tumor inoculation in mice showed complete inhibition of tumor growth, whereas administration of rE6mE7m did not. These results demonstrated that rlipo-E6mE7m could be used in tumors with E6 and/or E7 expression via the induction of E6- and E7-specific immunity.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6325468PMC
December 2018

Assessing the augmentation of Amblydromalus limonicus with the supplementation of pollen, thread, and substrates to combat greenhouse whitefly populations.

Sci Rep 2018 08 15;8(1):12189. Epub 2018 Aug 15.

School of Biological Sciences, The University of Auckland, Auckland, New Zealand.

Due to issues with establishment and persistence of natural enemies in biological control, the provision of alternative food sources and oviposition sites are important factors to enhance pest control. In this study, three different supplementation treatments were examined for their ability to increase the populations of the predatory mite Amblydromalus limonicus, and its implications for greenhouse whitefly control on peppers and eggplants. These were: (1) pollen (Typha orientalis), (2) pollen and thread, (3) pollen, thread, and a substrate mixture of buckwheat, gorse, and rice husks, which were compared to a control treatment that had no supplementation. Significant treatment effects were found on pepper for A. limonicus (mite eggs p = 0.008, mobile mites p = <0.0001). The predatory mite successfully established and persisted at high population levels in the pollen-thread, and pollen-thread-substrate treatments. All supplementation treatments were able to control whitefly populations on peppers, while the control treatment failed to. The results obtained were formulated into possible application techniques for greenhouse growers to utilise.
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http://dx.doi.org/10.1038/s41598-018-30018-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6093862PMC
August 2018

Real-Time Tumor Gene Expression Profiling to Direct Gastric Cancer Chemotherapy: Proof-of-Concept "3G" Trial.

Clin Cancer Res 2018 11 25;24(21):5272-5281. Epub 2018 Jul 25.

Department of Medicine, Yong Loo Lin School of Medicine, National University Health System, Singapore.

The oxaliplatin plus S-1 and cisplatin plus S-1 regimens are interchangeably used in the management of advanced gastric cancer. The previously reported G-intestinal (G1) and G-diffuse (G2) intrinsic gene expression signatures showed promise for stratifying patients according to their tumor sensitivity to oxaliplatin or cisplatin. The proof-of-concept, multicenter, open-label phase II "3G" trial was done to prospectively evaluate the feasibility and efficacy of using genomic classifiers to tailor treatment in gastric cancer. Patients' tumors were classified as "G1" or "G2" using a nearest-prediction template method, or "G3" (unclear assignment) when FDR ≥ 0.05. The first 30 patients in the "G1" cohort were assigned oxaliplatin plus S-1 (SOX) chemotherapy; thereafter, subsequently recruited "G1" patients were treated with cisplatin plus S-1 (SP) chemotherapy. "G2" patients and "G3" patients were treated with SP and SOX chemotherapy, respectively. A total of 48, 21, and 12 patients, respectively, were given "G1," "G2," and "G3" genomic assignments. Median turnaround time was 7 days (IQR, 5-9). Response rates were 44.8%, 8.3%, 26.7%, and 55.6% for the "G1-SOX," "G1-SP," "G2," "G3" cohorts, respectively; and was higher in G1 patients treated with SOX compared with SP ( = 0.033). Exploratory analyses using the genomic classifier of Lei and colleagues validated the utility of the metabolic signature as a biomarker for predicting benefit from chemotherapy (log-rank = 0.004 for PFS), whereas the Asian Cancer Research Group classifier did not demonstrate any predictive value. This bench-to-bedside effort establishes a reasonable turnaround time for gene expression profiling and possible utility of genomic classifiers in gastric cancer treatment stratification. .
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http://dx.doi.org/10.1158/1078-0432.CCR-18-0193DOI Listing
November 2018

Zfra restores memory deficits in Alzheimer's disease triple-transgenic mice by blocking aggregation of TRAPPC6AΔ, SH3GLB2, tau, and amyloid β, and inflammatory NF-κB activation.

Alzheimers Dement (N Y) 2017 Jun 6;3(2):189-204. Epub 2017 Mar 6.

Institute of Molecular Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan, ROC.

Introduction: Zinc finger-like protein that regulates apoptosis (Zfra) is a naturally occurring 31-amino-acid protein. Synthetic peptides Zfra1-31 and Zfra4-10 are known to effectively block the growth of many types of cancer cells.

Methods: Ten-month-old triple-transgenic (3×Tg) mice for Alzheimer's disease (AD) received synthetic Zfra peptides via tail vein injections, followed by examining restoration of memory deficits.

Results: Zfra significantly downregulated TRAPPC6AΔ, SH3GLB2, tau, and amyloid β (Αβ) aggregates in the brains of 3×Tg mice and effectively restored their memory capabilities. Zfra inhibited melanoma-induced neuronal death in the hippocampus and plaque formation in the cortex. Mechanistically, Zfra blocked the aggregation of amyloid β 42 and many serine-containing peptides in vitro, suppressed tumor necrosis factor-mediated NF-κB activation, and bound cytosolic proteins for accelerating their degradation in ubiquitin/proteasome-independent manner.

Discussion: Zfra peptides exhibit a strong efficacy in blocking tau aggregation and amyloid Αβ formation and restore memory deficits in 3×Tg mice, suggesting its potential for treatment of AD.
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http://dx.doi.org/10.1016/j.trci.2017.02.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5651433PMC
June 2017

Hyaluronan activates Hyal-2/WWOX/Smad4 signaling and causes bubbling cell death when the signaling complex is overexpressed.

Oncotarget 2017 Mar;8(12):19137-19155

Guthrie Research Institute, Laboratory of Molecular Immunology, Sayre, PA, USA.

Malignant cancer cells frequently secrete significant amounts of transforming growth factor beta (TGF-β), hyaluronan (HA) and hyaluronidases to facilitate metastasizing to target organs. In a non-canonical signaling, TGF-β binds membrane hyaluronidase Hyal-2 for recruiting tumor suppressors WWOX and Smad4, and the resulting Hyal-2/WWOX/Smad4 complex is accumulated in the nucleus to enhance SMAD-promoter dependent transcriptional activity. Yeast two-hybrid analysis showed that WWOX acts as a bridge to bind both Hyal-2 and Smad4. When WWOX-expressing cells were stimulated with high molecular weight HA, an increased formation of endogenous Hyal-2/WWOX/Smad4 complex occurred rapidly, followed by relocating to the nuclei in 20-40 min. In WWOX-deficient cells, HA failed to induce Smad2/3/4 relocation to the nucleus. To prove the signaling event, we designed a real time tri-molecular FRET analysis and revealed that HA induces the signaling pathway from ectopic Smad4 to WWOX and finally to p53, as well as from Smad4 to Hyal-2 and then to WWOX. An increased binding of the Smad4/Hyal-2/WWOX complex occurs with time in the nucleus that leads to bubbling cell death. In contrast, HA increases the binding of Smad4/WWOX/p53, which causes membrane blebbing but without cell death. In traumatic brain injury-induced neuronal death, the Hyal-2/WWOX complex was accumulated in the apoptotic nuclei of neurons in the rat brains in 24 hr post injury, as determined by immunoelectron microscopy. Together, HA activates the Hyal-2/WWOX/Smad4 signaling and causes bubbling cell death when the signaling complex is overexpressed.
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http://dx.doi.org/10.18632/oncotarget.13268DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5386674PMC
March 2017

Epigenomic profiling of primary gastric adenocarcinoma reveals super-enhancer heterogeneity.

Nat Commun 2016 Sep 28;7:12983. Epub 2016 Sep 28.

Cancer Therapeutics and Stratified Oncology, Genome Institute of Singapore, 60 Biopolis Street, Genome #02-01, Singapore 138672, Singapore.

Regulatory enhancer elements in solid tumours remain poorly characterized. Here we apply micro-scale chromatin profiling to survey the distal enhancer landscape of primary gastric adenocarcinoma (GC), a leading cause of global cancer mortality. Integrating 110 epigenomic profiles from primary GCs, normal gastric tissues and cell lines, we highlight 36,973 predicted enhancers and 3,759 predicted super-enhancers respectively. Cell-line-defined super-enhancers can be subclassified by their somatic alteration status into somatic gain, loss and unaltered categories, each displaying distinct epigenetic, transcriptional and pathway enrichments. Somatic gain super-enhancers are associated with complex chromatin interaction profiles, expression patterns correlated with patient outcome and dense co-occupancy of the transcription factors CDX2 and HNF4α. Somatic super-enhancers are also enriched in genetic risk SNPs associated with cancer predisposition. Our results reveal a genome-wide reprogramming of the GC enhancer and super-enhancer landscape during tumorigenesis, contributing to dysregulated local and regional cancer gene expression.
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http://dx.doi.org/10.1038/ncomms12983DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5052795PMC
September 2016

Upregulated, 7q21-22 amplicon candidate gene SHFM1 confers oncogenic advantage by suppressing p53 function in gastric cancer.

Cell Signal 2015 Jun 16;27(6):1075-86. Epub 2015 Feb 16.

Cancer Genetics Laboratory, Department of Genetics, Centre for Excellence in Genomic Sciences, School of Biological Sciences, Madurai Kamaraj University, Madurai, India. Electronic address:

Chromosomal aberrations are hallmarks of cancers and the locus of frequent genomic amplifications often harbors key cancer driver genes. Many genomic amplicons remain larger with hundreds of genes and the key drivers remain to be identified by an amplification-wide systematic analysis. The 7q21.12-q22.3 genomic amplification is frequent in gastric cancers which occur in ~10% of the patients and multiple cell lines. This 7q21.12-q22.3 amplicon has not yet been completely analyzed towards identifying the driver genes and their functional contribution in oncogenesis. The amplitude and prevalence indicate the important role conferred by this amplicon in gastric cancers. Among the 159 genes of this amplicon, 12 genes are found over-expressed in primary gastric tumors and cell lines. Many of the over-expressed genes show negative association with p53 transcriptional activity. RNAi based functional screening of the genes reveal, SHFM1 as key gastric cancer driver gene. SHFM1 confers cell cycle progression and resistance to p53 stabilizing drugs in gastric cancer cells. SHFM1 also activates Src, MAPK/ERK and PI3K/Akt signaling pathways. This is the first integrative genomic investigation of 7q21.12-q22.3 amplicon revealing the potential oncogenic candidacy of 12 genes. The oncogenic contribution of SHFM1, mediated by the p53 suppressive feature has been demonstrated in gastric cancer cells.
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http://dx.doi.org/10.1016/j.cellsig.2015.02.010DOI Listing
June 2015

Zfra activates memory Hyal-2+ CD3- CD19- spleen cells to block cancer growth, stemness, and metastasis in vivo.

Oncotarget 2015 Feb;6(6):3737-51

Institute of Molecular Medicine, National Cheng Kung University Medical College, Tainan, Taiwan, ROC.

Zfra is a 31-amino-acid zinc finger-like protein, which participates in the tumor necrosis factor signaling. Here, we determined that when nude mice and BALB/c mice were pre-injected with nanogram levels of a synthetic Zfra1-31 or truncated Zfra4-10 peptide via tail veins, these mice became resistant to the growth, metastasis and stemness of melanoma cells, and many malignant cancer cells. The synthetic peptides underwent self-polymerization in phosphate-buffered saline. Alteration of the Ser8 phosphorylation site to Gly8 abolished Zfra aggregation and its-mediated cancer suppression in vivo. Injected Zfra peptide autofluoresced due to polymerization and was trapped mainly in the spleen. Transfer of Zfra-stimulated spleen cells to naïve mice conferred resistance to cancer growth. Zfra-binding cells, designated Hyal-2+ CD3- CD19- Z cells, are approximately 25-30% in the normal spleen, but are significantly downregulated (near 0-3%) in tumor-growing mice. Zfra prevented the loss of Z cells caused by tumors. In vitro stimulation or education of naïve spleen cells with Zfra allowed generation of activated Z cells to confer a memory anticancer response in naïve or cancer-growing mice. In particular, Z cells are abundant in nude and NOD-SCID mice, and can be readily activated by Zfra to mount against cancer growth.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4414150PMC
http://dx.doi.org/10.18632/oncotarget.2895DOI Listing
February 2015

Trafficking protein particle complex 6A delta (TRAPPC6AΔ) is an extracellular plaque-forming protein in the brain.

Oncotarget 2015 Feb;6(6):3578-89

Institute of Molecular Medicine, National Cheng Kung University Medical College, Tainan, Taiwan, ROC.

Tumor suppressor WWOX is involved in the progression of cancer and neurodegeneration. Here, we examined whether protein aggregation occurs in the brain of nondemented, middle-aged humans and whether this is associated with WWOX downregulation. We isolated an N-terminal internal deletion isoform, TPC6AΔ, derived from alternative splicing of the TRAPPC6A (TPC6A) gene transcript. TPC6AΔ proteins are present as aggregates or plaques in the extracellular matrix of the brain such as in the cortex. Filter retardation assays revealed that aggregate formation of TPC6AΔ occurs preceding Aβ generation in the hippocampi of middle-aged postmortem normal humans. In a Wwox gene knockout mouse model, we showed the plaques of pT181-Tau and TPC6AΔ in the cortex and hippocampus in 3-week-old mice, suggesting a role of WWOX in limiting TPC6AΔ aggregation. To support this hypothesis, in vitro analysis revealed that TGF-β1 induces dissociation of the ectopic complex of TPC6AΔ and WWOX in cells, and then TPC6AΔ undergoes Ser35 phosphorylation-dependent polymerization and induces caspase 3 activation and Aβ production. Similarly, knockdown of WWOX by siRNA resulted in dramatic aggregation of TPC6AΔ. Together, when WWOX is downregulated, TPC6AΔ is phosphorylated at Ser35 and becomes aggregated for causing caspase activation that leads to Tau aggregation and Aβ formation.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4414138PMC
http://dx.doi.org/10.18632/oncotarget.2876DOI Listing
February 2015

Regulatory crosstalk between lineage-survival oncogenes KLF5, GATA4 and GATA6 cooperatively promotes gastric cancer development.

Gut 2015 May 22;64(5):707-19. Epub 2014 Jul 22.

Cancer and Stem Cell Biology program, Duke-NUS Graduate Medical School Singapore, Singapore, Singapore Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore Cellular and Molecular Research, National Cancer Centre, Singapore, Singapore Genome Institute of Singapore, Singapore, Singapore.

Objective: Gastric cancer (GC) is a deadly malignancy for which new therapeutic strategies are needed. Three transcription factors, KLF5, GATA4 and GATA6, have been previously reported to exhibit genomic amplification in GC. We sought to validate these findings, investigate how these factors function to promote GC, and identify potential treatment strategies for GCs harbouring these amplifications.

Design: KLF5, GATA4 and GATA6 copy number and gene expression was examined in multiple GC cohorts. Chromatin immunoprecipitation with DNA sequencing was used to identify KLF5/GATA4/GATA6 genomic binding sites in GC cell lines, and integrated with transcriptomics to highlight direct target genes. Phenotypical assays were conducted to assess the function of these factors in GC cell lines and xenografts in nude mice.

Results: KLF5, GATA4 and GATA6 amplifications were confirmed in independent GC cohorts. Although factor amplifications occurred in distinct sets of GCs, they exhibited significant mRNA coexpression in primary GCs, consistent with KLF5/GATA4/GATA6 cross-regulation. Chromatin immunoprecipitation with DNA sequencing revealed a large number of genomic sites co-occupied by KLF5 and GATA4/GATA6, primarily located at gene promoters and exhibiting higher binding strengths. KLF5 physically interacted with GATA factors, supporting KLF5/GATA4/GATA6 cooperative regulation on co-occupied genes. Depletion and overexpression of these factors, singly or in combination, reduced and promoted cancer proliferation, respectively, in vitro and in vivo. Among the KLF5/GATA4/GATA6 direct target genes relevant for cancer development, one target gene, HNF4α, was also required for GC proliferation and could be targeted by the antidiabetic drug metformin, revealing a therapeutic opportunity for KLF5/GATA4/GATA6 amplified GCs.

Conclusions: KLF5/GATA4/GATA6 may promote GC development by engaging in mutual crosstalk, collaborating to maintain a pro-oncogenic transcriptional regulatory network in GC cells.
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http://dx.doi.org/10.1136/gutjnl-2013-306596DOI Listing
May 2015

Nanoscale chromatin profiling of gastric adenocarcinoma reveals cancer-associated cryptic promoters and somatically acquired regulatory elements.

Nat Commun 2014 Jul 10;5:4361. Epub 2014 Jul 10.

1] Cancer Therapeutics and Stratified Oncology, Genome Institute of Singapore, 60 Biopolis Street, Genome #02-01, Singapore 138672, Singapore [2] Cancer and Stem Cell Biology Program, Duke-NUS Graduate Medical School, 8 College Road, Singapore 169857, Singapore [3] Cancer Science Institute of Singapore, National University of Singapore, 14 Medical Drive, #12-01, Singapore 117599, Singapore [4] Cellular and Molecular Research, National Cancer Centre, 11 Hospital Drive, Singapore 169610, Singapore.

Chromatin alterations are fundamental hallmarks of cancer. To study chromatin alterations in primary gastric adenocarcinomas, we perform nanoscale chromatin immunoprecipitation sequencing of multiple histone modifications in five gastric cancers and matched normal tissues. We identify hundreds of somatically altered promoters and predicted enhancers. Many cancer-associated promoters localize to genomic sites lacking previously annotated transcription start sites (cryptic promoters), driving expression of nearby genes involved in gastrointestinal cancer, embryonic development and tissue specification. Cancer-associated promoters overlap with embryonic stem cell regions targeted by polycomb repressive complex 2, exhibiting promoter bivalency and DNA methylation loss. We identify somatically acquired elements exhibiting germline allelic biases and non-coding somatic mutations creating new promoters. Our findings demonstrate the feasibility of profiling chromatin from solid tumours with limited tissue to identify regulatory elements, transcriptional patterns and regulatory genetic variants associated with cancer.
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http://dx.doi.org/10.1038/ncomms5361DOI Listing
July 2014

Genome-wide mutational signatures of aristolochic acid and its application as a screening tool.

Sci Transl Med 2013 Aug;5(197):197ra101

NCCS-VARI Translational Research Laboratory, Division of Medical Sciences, National Cancer Centre Singapore, 11 Hospital Drive, Singapore 169610, Singapore.

Aristolochic acid (AA), a natural product of Aristolochia plants found in herbal remedies and health supplements, is a group 1 carcinogen that can cause nephrotoxicity and upper urinary tract urothelial cell carcinoma (UTUC). Whole-genome and exome analysis of nine AA-associated UTUCs revealed a strikingly high somatic mutation rate (150 mutations/Mb), exceeding smoking-associated lung cancer (8 mutations/Mb) and ultraviolet radiation-associated melanoma (111 mutations/Mb). The AA-UTUC mutational signature was characterized by A:T to T:A transversions at the sequence motif A[C|T]AGG, located primarily on nontranscribed strands. AA-induced mutations were also significantly enriched at splice sites, suggesting a role for splice-site mutations in UTUC pathogenesis. RNA sequencing of AA-UTUC confirmed a general up-regulation of nonsense-mediated decay machinery components and aberrant splicing events associated with splice-site mutations. We observed a high frequency of somatic mutations in chromatin modifiers, particularly KDM6A, in AA-UTUC, demonstrated the sufficiency of AA to induce renal dysplasia in mice, and reproduced the AA mutational signature in experimentally treated human renal tubular cells. Finally, exploring other malignancies that were not known to be associated with AA, we screened 93 hepatocellular carcinoma genomes/exomes and identified AA-like mutational signatures in 11. Our study highlights an unusual genome-wide AA mutational signature and the potential use of mutation signatures as "molecular fingerprints" for interrogating high-throughput cancer genome data to infer previous carcinogen exposures.
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http://dx.doi.org/10.1126/scitranslmed.3006086DOI Listing
August 2013

Exome sequencing of liver fluke-associated cholangiocarcinoma.

Nat Genet 2012 May 6;44(6):690-3. Epub 2012 May 6.

National Cancer Centre Singapore-Van Andel Research Institute Translational Research Laboratory, Division of Medical Sciences, Singapore.

Opisthorchis viverrini-related cholangiocarcinoma (CCA), a fatal bile duct cancer, is a major public health concern in areas endemic for this parasite. We report here whole-exome sequencing of eight O. viverrini-related tumors and matched normal tissue. We identified and validated 206 somatic mutations in 187 genes using Sanger sequencing and selected 15 genes for mutation prevalence screening in an additional 46 individuals with CCA (cases). In addition to the known cancer-related genes TP53 (mutated in 44.4% of cases), KRAS (16.7%) and SMAD4 (16.7%), we identified somatic mutations in 10 newly implicated genes in 14.8-3.7% of cases. These included inactivating mutations in MLL3 (in 14.8% of cases), ROBO2 (9.3%), RNF43 (9.3%) and PEG3 (5.6%), and activating mutations in the GNAS oncogene (9.3%). These genes have functions that can be broadly grouped into three biological classes: (i) deactivation of histone modifiers, (ii) activation of G protein signaling and (iii) loss of genome stability. This study provides insight into the mutational landscape contributing to O. viverrini-related CCA.
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http://dx.doi.org/10.1038/ng.2273DOI Listing
May 2012

Identification of an In Vivo MEK/WOX1 Complex as a Master Switch for Apoptosis in T Cell Leukemia.

Genes Cancer 2011 May;2(5):550-62

Institute of Molecular Medicine, National Cheng Kung University Medical College, Tainan, Taiwan.

Not all leukemia T cells are susceptible to high levels of phorbol myristate acetate (PMA)-mediated apoptosis. At micromolar levels, PMA induces apoptosis of Jurkat T cells by causing mitochondrial polarization/de-polarization, release of cytosolic granules, and DNA fragmentation. Chemical inhibitors U0126 and PD98059 block mitogen-activated protein kinase kinase 1 (MEK1)-mediated phosphorylation of extracellular signal-regulated kinase (ERK) and prevent apoptosis. Mechanistically, proapoptotic tumor suppressor WOX1 (also named WWOX or FOR) physically interacts with MEK1, in part, in the lysosomes in Jurkat cells. PMA induces the dissociation, which leads to relocation of MEK1 to lipid rafts and WOX1 to the mitochondria for causing apoptosis. U0126 inhibits PMA-induced dissociation of WOX1/MEK1 complex and supports survival of Jurkat cells. In contrast, less differentiated Molt-4 T cells are resistant to PMA-induced dissociation of the WOX1/MEK1 complex and thereby are refractory to apoptosis. U0126 overturns the resistance for enhancing apoptosis in Molt-4 cells. Together, the in vivo MEK1/WOX1 complex is a master on/off switch for apoptosis in leukemia T cells.
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http://dx.doi.org/10.1177/1947601911418498DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3161421PMC
May 2011

Intrinsic subtypes of gastric cancer, based on gene expression pattern, predict survival and respond differently to chemotherapy.

Gastroenterology 2011 Aug 28;141(2):476-85, 485.e1-11. Epub 2011 Apr 28.

Department of Medical Oncology, National Cancer Centre Singapore, Singapore.

Background & Aims: Gastric cancer (GC) is a heterogeneous disease comprising multiple subtypes that have distinct biological properties and effects in patients. We sought to identify new, intrinsic subtypes of GC by gene expression analysis of a large panel of GC cell lines. We tested if these subtypes might be associated with differences in patient survival times and responses to various standard-of-care cytotoxic drugs.

Methods: We analyzed gene expression profiles for 37 GC cell lines to identify intrinsic GC subtypes. These subtypes were validated in primary tumors from 521 patients in 4 independent cohorts, where the subtypes were determined by either expression profiling or subtype-specific immunohistochemical markers (LGALS4, CDH17). In vitro sensitivity to 3 chemotherapy drugs (5-fluorouracil, cisplatin, oxaliplatin) was also assessed.

Results: Unsupervised cell line analysis identified 2 major intrinsic genomic subtypes (G-INT and G-DIF) that had distinct patterns of gene expression. The intrinsic subtypes, but not subtypes based on Lauren's histopathologic classification, were prognostic of survival, based on univariate and multivariate analysis in multiple patient cohorts. The G-INT cell lines were significantly more sensitive to 5-fluorouracil and oxaliplatin, but more resistant to cisplatin, than the G-DIF cell lines. In patients, intrinsic subtypes were associated with survival time following adjuvant, 5-fluorouracil-based therapy.

Conclusions: Intrinsic subtypes of GC, based on distinct patterns of expression, are associated with patient survival and response to chemotherapy. Classification of GC based on intrinsic subtypes might be used to determine prognosis and customize therapy.
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http://dx.doi.org/10.1053/j.gastro.2011.04.042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3152688PMC
August 2011

Genomic profiles specific to patient ethnicity in lung adenocarcinoma.

Clin Cancer Res 2011 Jun 26;17(11):3542-50. Epub 2011 Apr 26.

Genome Institute of Singapore, Genome, Singapore.

Purpose: East-Asian (EA) patients with non-small-cell lung cancer (NSCLC) are associated with a high proportion of nonsmoking women, epidermal growth factor receptor (EGFR)-activating somatic mutations, and clinical responses to tyrosine kinase inhibitors. We sought to identify novel molecular differences between NSCLCs from EA and Western European (WE) patients.

Experimental Design: A total of 226 lung adenocarcinoma samples from EA (n = 90) and WE (n = 136) patients were analyzed for copy number aberrations (CNA) by using a common high-resolution SNP (single nucleotide polymorphism) microarray platform. Univariate and multivariate analyses were carried out to identify CNAs specifically related to smoking history, EGFR mutation status, and ethnicity.

Results: The overall genomic profiles of adenocarcinomas from EA and WE patients were highly similar. Univariate analyses revealed several CNAs significantly associated with ethnicity, EGFR mutation, and smoking, but not to gender, and KRAS or p53 mutations. A multivariate model identified four ethnic-specific recurrent CNAs-significantly higher rates of copy number gain were observed on 16p13.13 and 16p13.11 in EA tumors, whereas higher rates of genomic loss on 19p13.3 and 19p13.11 were observed in tumors from WE patients. We identified several potential driver genes in these regions, showing a positive correlation between cis-localized copy number changes and transcriptomic changes.

Conclusion: 16p copy number gains (EA) and 19p losses (WE) are ethnic-specific chromosomal aberrations in lung adenocarcinoma. Patient ethnicity should be considered when evaluating future NSCLC therapies targeting genes located on these areas.
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http://dx.doi.org/10.1158/1078-0432.CCR-10-2185DOI Listing
June 2011

Zfra is a small wizard in the mitochondrial apoptosis.

Aging (Albany NY) 2010 Dec;2(12):1023-9

Institute of Molecular Medicine, National Cheng Kung University Medical College, Tainan, Taiwan, ROC.

Zfra (zinc finger-like protein that regulates apoptosis) is a naturally occurring short peptide consisting of 31 amino acids, which regulates tumor necrosis factor (TNF)-mediated cell death by interacting with receptor adaptor protein TRADD (TNF receptorassociated death domain protein) and downstream JNK (c-Jun N-terminal kinase), NF-κB (Nuclear factor kappa B) and WWOX/WOX1 (WW domain-containing oxidoreductase). Cytochrome c release is generally considered as a pivotal step in apoptosis. Remarkably, overexpressed Zfra induces apoptosis via the mitochondrial pathway, which involves suppression of Bcl-2 and Bcl-xL expression (without causing cytochrome c release), counteracting the apoptotic function of tumor suppressor p53 and WWOX, and dissipation of mitochondrial membrane potential for ultimately leading to cell death. Control of cellular aging and apoptosis by Zfra, p53 and WWOX is discussed.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3034171PMC
http://dx.doi.org/10.18632/aging.100263DOI Listing
December 2010

Dramatic co-activation of WWOX/WOX1 with CREB and NF-kappaB in delayed loss of small dorsal root ganglion neurons upon sciatic nerve transection in rats.

PLoS One 2009 Nov 12;4(11):e7820. Epub 2009 Nov 12.

Department of Cell Biology & Anatomy, National Cheng Kung University Medical College, Tainan, Taiwan.

Background: Tumor suppressor WOX1 (also named WWOX or FOR) is known to participate in neuronal apoptosis in vivo. Here, we investigated the functional role of WOX1 and transcription factors in the delayed loss of axotomized neurons in dorsal root ganglia (DRG) in rats.

Methodology/principal Findings: Sciatic nerve transection in rats rapidly induced JNK1 activation and upregulation of mRNA and protein expression of WOX1 in the injured DRG neurons in 30 min. Accumulation of p-WOX1, p-JNK1, p-CREB, p-c-Jun, NF-kappaB and ATF3 in the nuclei of injured neurons took place within hours or the first week of injury. At the second month, dramatic nuclear accumulation of WOX1 with CREB (>65% neurons) and NF-kappaB (40-65%) occurred essentially in small DRG neurons, followed by apoptosis at later months. WOX1 physically interacted with CREB most strongly in the nuclei as determined by FRET analysis. Immunoelectron microscopy revealed the complex formation of p-WOX1 with p-CREB and p-c-Jun in vivo. WOX1 blocked the prosurvival CREB-, CRE-, and AP-1-mediated promoter activation in vitro. In contrast, WOX1 enhanced promoter activation governed by c-Jun, Elk-1 and NF-kappaB. WOX1 directly activated NF-kappaB-regulated promoter via its WW domains. Smad4 and p53 were not involved in the delayed loss of small DRG neurons.

Conclusions/significance: Rapid activation of JNK1 and WOX1 during the acute phase of injury is critical in determining neuronal survival or death, as both proteins functionally antagonize. In the chronic phase, concurrent activation of WOX1, CREB, and NF-kappaB occurs in small neurons just prior to apoptosis. Likely in vivo interactions are: 1) WOX1 inhibits the neuroprotective CREB, which leads to eventual neuronal death, and 2) WOX1 enhances NF-kappaB promoter activation (which turns to be proapoptotic). Evidently, WOX1 is the potential target for drug intervention in mitigating symptoms associated with neuronal injury.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0007820PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2771921PMC
November 2009

Complement C1q activates tumor suppressor WWOX to induce apoptosis in prostate cancer cells.

PLoS One 2009 Jun 1;4(6):e5755. Epub 2009 Jun 1.

Guthrie Research Institute, Laboratory of Molecular Immunology, Sayre, PA, USA.

Background: Tissue exudates contain low levels of serum complement proteins, and their regulatory effects on prostate cancer progression are largely unknown. We examined specific serum complement components in coordinating the activation of tumor suppressors p53 and WWOX (also named FOR or WOX1) and kinases ERK, JNK1 and STAT3 in human prostate DU145 cells.

Methodology/principal Findings: DU145 cells were cultured overnight in 1% normal human serum, or in human serum depleted of an indicated complement protein. Under complement C1q- or C6-free conditions, WOX1 and ERK were mainly present in the cytoplasm without phosphorylation, whereas phosphorylated JNK1 was greatly accumulated in the nuclei. Exogenous C1q rapidly restored the WOX1 activation (with Tyr33 phosphorylation) in less than 2 hr. Without serum complement C9, p53 became activated, and hyaluronan (HA) reversed the effect. Under C6-free conditions, HA induced activation of STAT3, an enhancer of metastasis. Notably, exogenous C1q significantly induced apoptosis of WOX1-overexpressing DU145 cells, but not vehicle-expressing cells. A dominant negative and Y33R mutant of WOX1 blocked the apoptotic effect. C1q did not enhance p53-mediated apoptosis. By total internal reflection fluorescence (TIRF) microscopy, it was determined that C1q destabilized adherence of WOX1-expressing DU145 cells by partial detaching and inducing formation of clustered microvilli for focal adhesion particularly in between cells. These cells then underwent shrinkage, membrane blebbing and death. Remarkably, as determined by immunostaining, benign prostatic hyperplasia and prostate cancer were shown to have a significantly reduced expression of tissue C1q, compared to age-matched normal prostate tissues.

Conclusions/significance: We conclude that complement C1q may induce apoptosis of prostate cancer cells by activating WOX1 and destabilizing cell adhesion. Downregulation of C1q enhances prostate hyperplasia and cancerous formation due to failure of WOX1 activation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0005755PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2685983PMC
June 2009