Effectiveness of early palliative care in patients with head and neck cancer in Taiwan.

BACKGROUND: Early palliative care (EPC) benefits some cancers, but its clinical outcomes differ depending on patients' racial and ethnic disparities, and customs. To determine whether EPC improves symptoms, emotional distress, and quality of life among Taiwanese patients with early or advanced-stage head and neck cancer (HNC). METHODS: Based on participants' pathological stages, they were categorized as having early and advanced-stage HNC. Those willing and unwilling to undergo EPC were assigned to the EPC and standard groups, respectively. Their daily cancer-related symptoms were assessed using the Distress Thermometer (DT) and MD Anderson Symptom Inventory (MDASI), whose scores' concurrent validity was evaluated using the European Organization for Research and Treatment of Core Quality of Life (EORTC-QLQ-C30) and Head and Neck 35 (EORTC-QLQ-H&N35) questionnaires. RESULTS: Patients (n = 93) diagnosed with HNC at Taiwan's Chia-Yi Christian Hospital from November 2020 to October 2022 were recruited. The patients voluntarily split into two groups: EPC groups and standard groups (23 and 11 in early-stage; 46 and 13 in advanced-stage, respectively). DT assessment showed significant emotional distress improvements for all patients with HNC who received EPC. The EORTC-QLQ-C30 questionnaire indicated that, compared to standard interventions, EPC groups significantly improved the quality of life and some symptoms for both early and advanced-stage HNC patients. However, the EORTC-QLQ-H&N35 questionnaire found no significant difference between the two groups. Furthermore, advanced-stage patients' anticancer treatment completion rates with EPC and standard interventions were 95.35% and 75%, respectively. CONCLUSION: EPC improves symptoms, emotional distress, quality of life, and treatment completion rates in Taiwanese patients with early or advanced-stage HNC. Nonetheless, further extensive clinical studies are required for validation.

Nutraceutical Potential of Djulis (Chenopodium formosanum) Hull: Phytochemicals, Antioxidant Activity, and Liver Protection.

Djulis (Chenopodium formosanum), a traditional Taiwanese crop enriched with phenolic compounds and betalain pigments, is associated with various health benefits, including antioxidant and hepatoprotective effects. This study analysed the phytochemical content and antioxidant capacity of extracts from both the hull and kernel of Djulis. The hull extract, which contained higher levels of flavonoids and exhibited superior antioxidant activity compared to the kernel extract, was selected for further in vivo studies. These experiments showed that oral administration of the Djulis hull crude extract significantly mitigated lipopolysaccharide (LPS)-induced acute liver injury (ALI) in mice by increasing the activity of the antioxidant enzyme glutathione peroxidase (GPx), reducing plasma levels of pro-inflammatory cytokine interferon gamma (IFN-γ), and enhancing liver levels of the anti-inflammatory cytokine interleukin-4 (IL-4). Additionally, the extract demonstrated potential in inhibiting the TLR4/NF-κB pathway, a critical signalling pathway in inflammation and apoptosis, offering insights into its protective mechanisms. These findings underscore Djulis hull's potential as a functional food ingredient for ALI prevention and propose a valuable application for agricultural by-products.

Increased Apolipoprotein A1 Expression Correlates with Tumor-Associated Neutrophils and T Lymphocytes in Upper Tract Urothelial Carcinoma.

An increased neutrophil-to-lymphocyte ratio (NLR) is a poor prognostic biomarker in various types of cancer, because it reflects the inhibition of lymphocytes in the circulation and tumors. In urologic cancers, upper tract urothelial carcinoma (UTUC) is known for its aggressive features and lack of T cell infiltration; however, the association between neutrophils and suppressed T lymphocytes in UTUC is largely unknown. In this study, we examined the relationship between UTUC-derived factors and tumor-associated neutrophils or T lymphocytes. The culture supernatant from UTUC tumor tissue modulated neutrophils to inhibit T cell proliferation. Among the dominant factors secreted by UTUC tumor tissue, apolipoprotein A1 (Apo-A1) exhibited a positive correlation with NLR. Moreover, tumor-infiltrating neutrophils were inversely correlated with tumor-infiltrating T cells. Elevated Apo-A1 levels in UTUC were also inversely associated with the population of tumor-infiltrating T cells. Our findings indicate that elevated Apo-A1 expression in UTUC correlates with tumor-associated neutrophils and T cells. This suggests a potential immunomodulatory effect on neutrophils and T cells within the tumor microenvironment, which may represent therapeutic targets for UTUC treatment.

Anti-Inflammatory Effects of Anthocyanin-Enriched Black Soybean Seed Coat (BSSC) Crude Extract on LPS-Induced Acute Liver Injury in Mice.

Soybeans rank among the top five globally produced crops. Black soybeans contain anthocyanins in their seed coat, offering strong antioxidant and anti-inflammatory benefits. This study explores the protective effects of black soybean seed coat (BSSC) against acute liver injury (ALI) in mice. Mice pretreated with BSSC crude extract showed reduced liver damage, inflammation, and apoptosis. High doses (300 mg/kg) of the extract decreased levels of proinflammatory cytokines (IL-6, IFN-γ) and increased levels of anti-inflammatory ones (IL-4, IL-10), alongside mitigating liver pathological damage. Additionally, it influenced the Nrf2/HO-1 pathway and reduced levels of apoptosis-related proteins. In vitro, the compounds delphinidin-3-O-glucoside (D3G) and cyanidin-3-O-glucoside (C3G) in BSSC were found to modulate cytokine levels, suggesting their role in ALI protection. The study concludes that BSSC extract, particularly due to D3G and C3G, effectively protects against LPS-induced ALI in mice by inhibiting inflammation, oxidative stress, and apoptosis.

Tonsil tissue control is ideal for monitoring estrogen receptor immunohistochemical staining.

BACKGROUND: Estrogen receptor (ER) testing performed using immunohistochemistry (IHC) is a critical predictive tool for breast cancer treatment. This study aimed to investigate the use of tonsil control for monitoring ER staining and hypothesize that optimal staining would reduce interlaboratory variations. METHODS: A proficiency test for ER IHC was conducted using 21 tissue cores. The staining quality was centrally reviewed based on tonsil ER staining. RESULTS: We found that 64.9% of participant samples demonstrated optimal or good staining quality. Poor staining quality was significantly associated with the use of Ventana autostainers and concentrated antibodies. Although the concordance rate did not show significant differences across staining quality levels, interparticipant agreement declined as staining quality deteriorated. Among the 19 discordant responses, 63.2% could be attributed to staining problems, whereas 36.8% could be due to misinterpretation. Poor staining quality due to inadequate staining was the primary reason for undercalls, which can lead to false-negative results. Misinterpretations of nonspecific faint staining that was weaker than the staining of the tonsil control were the cause of most overcalls. CONCLUSION: Tonsil tissue is an ideal control for monitoring ER staining and can serve as a reference for determining the lower bound for ER positivity. Optimal ER staining and appropriate references for ER positivity can further improve ER IHC quality.

Suppression of bone metastatic castration-resistant prostate cancer cell growth by a suicide gene delivered by JC polyomavirus-like particles.

Prostate cancer is one of the most common cancers in men. The heterogeneity and mutations exhibited by prostate cancer cells often results in the progression to incurable metastatic castration-resistant prostate cancer (mCRPC). Our previous investigations demonstrated that the virus-like particles (VLPs) of JC polyomavirus (JCPyV) can deliver exogenous genes to prostate cancer cells for expression. JCPyV VLPs packaging pPSAtk (PSAtk-VLPs) possess the ability to transcriptionally target and selectively induce cytotoxicity in prostate cancer cells in vitro and in vivo, as pPSAtk can only express the thymidine kinase gene, a suicide gene, in androgen receptor-positive cells. To further investigate whether PSAtk-VLPs inhibit the growth of metastasized prostate cancer cells, we established an animal model of bone-metastatic prostate cancer to compare PSAtk-VLPs with leuprorelin acetate and enzalutamide, hormonal agents commonly used in clinical settings, and investigated the effectiveness of PSAtk-VLPs. In the present study, we observed that PSAtk-VLPs effectively inhibited the growth of prostate cancer cells that had metastasized to the bone in the metastatic animal model. In addition, PSAtk-VLPs showed a higher effectiveness than hormone therapy in this animal model study. These results suggest that PSAtk-VLPs may serve as a treatment option for mCRPC therapy in the future.

Ivermectin induces cell cycle arrest and caspase-dependent apoptosis in human urothelial carcinoma cells.

Bladder carcinoma is one of the most common malignancies worldwide, and >90% of all bladder cancers are classified as urothelial carcinomas (UC). Surgery, radiotherapy, chemotherapy, targeted therapy, and immunotherapy are evidence-based treatments that are administered depending on the clinical stage of UC. All these treatments exhibited limited effects in cases of metastatic UC, and UC with specific location, invasiveness, and recurrence. Therefore, a new therapeutic strategy for UC is urgently needed. Ivermectin, an avermectin derivative, has been reported to be effective against various parasites, and its pharmacokinetic and pharmacodynamic properties as well as safety are well understood in humans. Recently, ivermectin was shown to exhibit therapeutic benefits against various virus infections in vitro, and anticancer activity against various human cancer cells. This study aimed to investigate the anticancer effects of ivermectin in human UC cells. Ivermectin inhibited growth, regulated the cell cycle, and induced apoptosis in human UC cells. It also induced the activation of both extrinsic and intrinsic caspase-dependent apoptotic pathways. Further investigation revealed that ivermectin induced apoptosis in UC cells is mediated via c-Jun N-terminal kinase signaling. Herein, we demonstrated that ivermectin can be used as a new therapeutic agent for treating UC cells.

Microbiome dysbiosis inhibits carcinogen-induced murine oral tumorigenesis.

Oral cancer is one of the most common cancers worldwide and ranks fourth for the mortality rate of cancers in males in Taiwan. The oral microbiota is the microbial community in the oral cavity, which is essential for maintaining oral health, but the relationship between oral tumorigenesis and the oral microbiota remains to be clarified. This study evaluated the effect of microbiome dysbiosis on oral carcinogenesis in mice, and the impact of the microbiome and its metabolic pathways on regulating oral carcinogenesis. We found that antibiotics treatment decreases carcinogen-induced oral epithelial malignant transformation. Microbiome analysis based on 16S rRNA gene sequencing revealed that the species richness of fecal specimens was significantly reduced in antibiotic-treated mice, while that in the salivary specimens was not decreased accordingly. Differences in bacterial composition, including Lactobacillus animalis abundance, in the salivary samples of cancer-bearing mice was dramatically decreased. L. animalis was the bacterial species that increased the most in the saliva of antibiotic-treated mice, suggesting that L. animalis may be negatively associated with oral carcinogenesis. In functional analysis, the microbiome in the saliva of the tumor-bearing group showed greater potential for polyamine biosynthesis. Immunochemical staining proved that spermine oxidase, an effective polyamine oxidase, was upregulated in mouse oral cancer lesions. In conclusion, oral microbiome dysbiosis may alter polyamine metabolic pathways and reduce carcinogen-induced malignant transformation of the oral epithelium.

Unusually high incidence of polyomavirus JC infection in the higher grade of colorectal cancer tissues in Taiwan.

INTRODUCTION: The human JC polyomavirus (JCPyV) has been detected in colorectal cancer (CRC) tissues and is suggested to contribute to CRC tumorigenesis. The rearrangement of the JCPyV regulatory region is supposedly associated with CRC development. The progression of CRC involves the stepwise accumulation of mutations. The large tumor antigen (LT) of JCPyV can trigger uncontrolled cell cycle progression by targeting oncogenes, and tumor suppressor genes, and causing chromosome instability. Few studies have focused on the presence of JCPyV DNA in the higher grade of CRC tissues. METHODS: We collected 95 tissue blocks from samples of stages I, II, III, and IV CRC. Nested PCR targeting the regulatory region of the viral genome was performed to determine the presence of JCPyV DNA in the various stages of colorectal cancer tissues. RESULTS: The nested PCR results showed that the positive rate of JCPyV DNA increased with the progression of CRC stages. The archetypal-like, non-rearrangement genotype of JCPyV with subtle mutations was the major genotype found in CRC samples. CONCLUSIONS: This finding in our study suggests that there may be an association between JCPyV and CRC progression.

Identifying pathological slices of gastric cancer via deep learning.

BACKGROUND: The accuracy of histopathology diagnosis largely depends on the pathologist's experience. It usually takes over 10 years to cultivate a senior pathologist, and small numbers of them lead to a high workload for those available. Meanwhile, inconsistent diagnostic results may arise among different pathologists, especially in complex cases, because diagnosis based on morphology is subjective. Computerized analysis based on deep learning has shown potential benefits as a diagnostic strategy. METHODS: This research aims to automatically determine the location of gastric cancer (GC) in the images of GC slices through artificial intelligence. We use image data from a regional teaching hospital in Taiwan for training. We collect images of patients diagnosed with GC from January 1, 2019 to December 31, 2020. In this study, scanned images are used to dissect 13,600 images from 50 different patients with GC sections whose GC sections are stained with hematoxylin and eosin (H&E stained) through a whole slide scanner, the scanned images from 50 different GC slice patients are divided into 80% training combinations, 2200 images of 40 patients are trained. The remaining 20%, totaling 10 people, are validated from a test set of 550 images. RESULTS: The validation results show that 91% of the correct rates are interpreted as GC images through deep learning. The sensitivity, specificity, PPV, and NPV were 84.9%, 94%, 87.7%, and 92.5%, respectively. After creating a 3D model through the grayscale value, the position of the GC is completely marked by the 3D model. The purpose of this research is to use artificial intelligence (AI) to determine the location of the GC in the image of GC slice. CONCLUSION: In patients undergoing pancreatectomy for pancreatic cancer, intraoperative infusion of lidocaine did not improve overall or disease-free survival. Reduced formation of circulating NETs was absent in pancreatic tumour tissue. CONCLUSION: For AI to assist pathologists in daily practice, to help a pathologist making a definite diagnosis is not the prime purpose at present time. The benefits could come from cancer screening and double-check quality control in a heavy workload which could distract the attention of pathologist during the time constraint examination process. We propose a two-steps method to identify cancerous areas in endoscopic gastric biopsy slices via deep learning. Then a 3D model is used to further mark all the positions of GC in the picture, and the model overcomes the problem that deep learning can't catch all GC.

Endurance exercise ameliorates Western diet-induced atherosclerosis through modulation of microbiota and its metabolites.

The World Health Organization determined cardiovascular disease to be the leading cause of death globally; atherosclerosis is the primary cause of the high morbidity and mortality rates. Regular physical activity is an effective strategy for maintaining endothelial health and function to prevent the development of atherosclerosis. Obesity is also a crucial risk factor for atherosclerotic progression in combination with various complications and systemic inflammation. Physiological homeostasis is modulated by the intestinal microbiota, but the mechanisms through which exercise attenuates atherosclerosis through the microbiota have not been elucidated. Therefore, we investigated the effects of endurance exercise on atherosclerosis induced by a Western diet (WD) and apolipoprotein E (ApoE) knockout in terms of microbiota parameters and metabolites. Genetically modified ApoE knockout mice (C57BL/6-Apoeem1Narl/Narl, ApoEKO) and wild-type mice (C57BL6/J) were divided into the following four groups (n = 6), namely, wild-type mice fed a chow diet (WT CD), ApoEKO mice fed a chow diet (ApoE CD), ApoEKO mice fed a WD (ApoE WD), and ApoEKO mice fed a WD and performing endurance exercise (ApoE WD EX), for a 12-week intervention. The WD significantly induced obesity and atherosclerotic syndrome in the ApoE WD group. Severe atherosclerotic lesions and arterial thickness were significantly elevated and accompanied by increases in VCAM-1, MCP-1, TNF-α, and IL-1ß for immune cell chemotaxis and inflammation during atherosclerotic pathogenesis in the ApoE WD group. In addition, dysbiosis in the ApoE WD group resulted in the lowest short-chain fatty acid (SCFA) production. Endurance exercise intervention (ApoE WD EX) significantly alleviated atherosclerotic syndrome by reducing obesity, significantly inhibiting VCAM-1, MCP-1, TNF-α, and IL-1ß expression, and increasing the production of SCFAs. Modulation of the microbiota associated with inflammation, such as Desulfovibrio, Tyzzerella, and Lachnospiraceae_ge, and increased SCFA production, particularly through an abundance of Rikenellaceae and Dubosiella, were also observed after exercise intervention. Endurance exercise can alleviate WD-induced atherosclerosis through the amelioration of obesity, inflammation, and chemotaxis signaling, which are modulated by the microbiota and derived SCFAs.

Proteomic Analysis of Metastasis-Specific Biomarkers in Pancreatic Cancer: Galectin-1 Plays an Important Metastatic Role in Pancreatic Cancer.

Cancer metastasis is the major cause of death in pancreatic cancer. We have established a pair of pancreatic ductal adenocarcinoma cell line, PANC1 and invasive PANC1-I5, as a model system toinvestigate the metastatic mechanism as well as potential therapeutic targets in pancreatic cancer. We used proteomic analysis based on two-dimensional differential gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) to examine the global protein expression alterations between PANC1 and PANC1-I5. Proteomic study revealed that 88 proteins are differentially expressed between PANC1-I5 and PANC1 cells, and further functional evaluations through protein expression validation, gene knockout, migration and invasion analysis revealed that galectin-1 is one of the potential players in modulating pancreatic cancer metastasis. To conclude, we have identified numerous proteins might be associated with pancreatic cancer invasiveness in the pancreatic cancer model.

Carthamus tinctorius L. extract activates insulin-like growth factor-I receptor signaling to inhibit FAS-death receptor pathway and suppress lipopolysaccharides-induced H9c2 cardiomyoblast cell apoptosis.

Carthamus tinctorius L. (Compositae) is used in Chinese medicine to treat heart disease and inflammation. In our previous study, we found that C. tinctorius L. inhibited lipopolysaccharides (LPS)-induced tumor necrosis factor-alpha (TNF-α) activation, JNK expression, and apoptosis in H9c2 cardiomyoblast cells. The present study was performed to investigate the protective effect of C. tinctorius extract (CTF) on LPS-challenged H9c2 myocardioblast cell and to explore the possible underlying mechanism. Cell viability assay showed that LPS treatment decreased the cell viability of H9c2 cell, whereas CTF treatment reversed LPS cytotoxicity in a dose-dependent manner, especially in the LPS + CTF 25 (µg/mL) group. LPS treatment-induced apoptosis was determined by transferase-mediated dUTP nick end labeling assay, and by Western blot. LPS-induced apoptotic bodies were decreased following CTF treatment. Expression of TNF-α, FAS-L, FAS, FADD, caspase-8, BID, and t-BID was significantly increased in LPS-treated H9c2 cells. In contrast, it was significantly suppressed by the administration of CTF extract. In addition, CTF treatment activates antiapoptotic proteins, Bcl-2 and p-Bad, and downregulates Bax, cytochrome-c, caspase-9, caspase-3, and apoptosis-inducing factor expression. Furthermore, CTF exerted cytoprotective effects by activating insulin-like growth factor-I (IGF-I) signaling pathway leading to downregulation of the apoptotic proteins involved in FAS death receptor pathway. In addition, AG1024 and IGF-I receptor (IGF-IR) inhibitor and siRNA silencing reverses the effect of CTF implying that CTF functions through the IGF-IR pathway to inhibit LPS-induced H9c2 apoptosis. These results suggest that treatment with CTF extract prevented the LPS-induced apoptotic response through IGF-I pathway.

Salinomycin acts through reducing AKT-dependent thymidylate synthase expression to enhance erlotinib-induced cytotoxicity in human lung cancer cells.

Erlotinib (TarcevaR) is a selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor in the treatment of human non-small cell lung cancer (NSCLC). Salinomycin, a polyether antibiotic, has been promising a novel therapeutic agent for lung cancer, and down-regulated the expression of thymidylate synthase (TS) in NSCLC cell lines. Previous study showed that against EGFR and TS was strongly synergistic cytotoxicity in NSCLC cells. In this study, we showed that erlotinib (1.25-10µM) treatment down-regulating of TS expression in an AKT inactivation manner in two NSCLC cell lines, human lung squamous cell carcinoma H1703 and adenocarcinoma H1975 cells. Knockdown of TS using small interfering RNA (siRNA) or inhibiting AKT activity with PI3K inhibitor LY294002 enhanced the cytotoxicity and cell growth inhibition of erlotinib. A combination of erlotinib and salinomycin resulted in synergistic enhancement of cytotoxicity and cell growth inhibition in NSCLC cells, accompanied with reduced protein levels of phospho-AKT(Ser473), phospho-AKT(Thr308), and TS. Overexpression of a constitutive active AKT (AKT-CA) or Flag-TS expression vector reversed the salinomycin and erlotinib-induced synergistic cytotoxicity. Our findings suggested that the down-regulation of AKT-mediated TS expression by salinomycin enhanced the erlotinib-induced cytotoxicity in NSCLC cells. These results may provide a rationale to combine salinomycin with erlotinib for lung cancer treatment.

18F-FDG PET/CT of Seminal Vesicle Metastasis From Ascending Colon Adenocarcinoma.

The most common metastatic sites of colorectal cancer are the regional lymph nodes, liver, lungs, and peritoneum. Seminal vesicle metastasis from colon adenocarcinoma is not reported yet; the diagnosis could be challenging. We report a case of seminal vesicle metastasis from a primary ascending colon adenocarcinoma in a 49-year-old man, with elevating carcinoembryonic antigen as the only clinical sign. This also highlights the role of F-FDG PET/CT to detect metastatic adenocarcinoma from colon in the genitourinary system.

Foxp3 enhances HIF-1α target gene expression in human bladder cancer through decreasing its ubiquitin-proteasomal degradation.

Hypoxia-inducible factor-1α (HIF-1α) can control a transcriptional factor forkhead box P3 (Foxp3) protein expression in T lymphocyte differentiation through proteasome-mediated degradation. In this study, we unveil a reverse regulatory mechanism contributing to bladder cancer progression; Foxp3 expression attenuates HIF-1α degradation. We first demonstrated that Foxp3 expression positively correlates with the metastatic potential in T24 cells and can increase the expression of HIF-1α-target genes, such as vascular endothelial growth factor (VEGF) and glucose transporter (GLUT). Foxp3 protein can bind with HIF-1α, particularly under hypoxia. In vivo ubiquination assay demonstrated that Foxp3 can decrease HIF-1α degradation in a dose-dependent manner. Knocking-down of Foxp3 expression blocks in vivo tumor growth in mice and prolongs mice's survival, which is associated with von Willebrand factor expression. Thirty-three of 145 (22.8 %) bladder tumors exhibit Foxp3 expression. Foxp3 expression is an independent predictor for disease progression in superficial bladder cancer patients (p = 0.032), associated with less number of intratumoral CD8+ lymphocyte. The metaanalysis from 2 published datasets showed Foxp3 expression is positively associated with GLUT-4,-9, and VEGF-A, B-, D expression. This reverse post-translational regulation of HIF-1α protein by Foxp3 provides a new potential target for developing new therapeutic strategy for bladder cancer.

Curcumin downregulates p38 MAPK-dependent X-ray repair cross-complement group 1 (XRCC1) expression to enhance cisplatin-induced cytotoxicity in human lung cancer cells.

Cisplatin is a well-studied and widely used chemotherapeutic agent and is effective in the treatment of the advanced human non-small cell lung cancer (NSCLC). Curcumin is a yellow pigment derived from the rhizome of Curcuma longa and has been proved to have antioxidant and antitumor properties. XRCC1 is an important scaffold protein involved in base excision repair and plays an important role in the development of lung cancer. In this study, we characterize the role of curcumin in the cytotoxicity, p38 MAPK activation, and XRCC1 expression affected by cisplatin in NSCLC cells. We show that curcumin enhanced the cytotoxicity induced by cisplatin in two NSCLC cells, A549 and H1703. Treatment with cisplatin alone increased XRCC1 mRNA and protein expression through p38 MAPK activation. Moreover, SB2023580 (p38 inhibitor) decreased the XRCC1 mRNA and protein stability upon cisplatin treatment. Knockdown of XRCC1 in NSCLC cells by transfection of XRCC1 siRNA or inactivation of p38 MAPK resulted in enhancing the cytotoxicity and cell growth inhibition induced by cisplatin. Curcumin inhibited the expression of XRCC1 in cisplatin-exposed NSCLC cells. Furthermore, transfection with constitutive active MKK6 or HA-p38 MAPK vectors rescued the XRCC1 protein level and also the cell survival suppressed by cisplatin and curcumin combination in A549 and H1703 cells. These findings suggested that the downregulation of XRCC1 expression by curcumin can enhance the chemosensitivity of cisplatin in NSCLC cells.

Methylomics analysis identifies ZNF671 as an epigenetically repressed novel tumor suppressor and a potential non-invasive biomarker for the detection of urothelial carcinoma.

The molecular mechanism underlying the lethal phenomenon of urothelial carcinoma (UC) tumor recurrence remains unresolved. Here, by methylation microarray, we identified promoter methylation of the zinc-finger protein gene, ZNF671 in bladder UC tumor tissue samples, a finding that was independently validated by bisulphite pyrosequencing in cell lines and tissue samples. Subsequent assays including treatment with epigenetic depressive agents and in vitro methylation showed ZNF671 methylation to result in its transcriptional repression. ZNF671 re-expression in UC cell lines, via ectopic expression, inhibited tumor growth and invasion, in possible conjunction with downregulation of cancer stem cell markers (c-KIT, NANOG, OCT4). Clinically, high ZNF671 methylation in UC tumor tissues (n=96; 63 bladder, 33 upper urinary tract) associated with tumor grade and poor locoregional disease-free survival. Quantitative MSP analysis in a training (n=97) and test (n=61) sets of voided urine samples from bladder UC patients revealed a sensitivity and specificity of 42%-48% and 89%-92.8%, respectively, for UC cancer detection. Moreover, combining DNA methylation of ZNF671 and 2 other genes (IRF8 and sFRP1) further increased the sensitivity to 96.2%, suggesting a possible three-gene UC biomarker. In summary, ZNF671, an epigenetically silenced novel tumor suppressor, represents a potential predictor for UC relapse and non-invasive biomarker that could assist in UC clinical decision-making.