Search

Lupus nephritis (LN) leads to chronic kidney disease (CKD) through progressive fibrosis. Mycophenolate inhibits inosine monophosphate dehydrogenase and is a standard treatment for LN. The mammalian or mechanistic target of rapamycin (mTOR) pathway is activated in LN. Rapamycin inhibits mTOR and is effective in preventing kidney transplant rejection, with the additional merits of reduced incidence of malignancies and viral infections. The effect of mycophenolate or rapamycin on kidney fibrosis in LN has not been investigated. We investigated the effects of mycophenolate and rapamycin in New Zealand Black and White first generation (NZB/W F1) murine LN and human mesangial cells (HMCs), focusing on mechanisms leading to kidney fibrosis. Treatment of mice with mycophenolate or rapamycin improved nephritis manifestations, decreased anti-double stranded (ds) DNA antibody titer and reduced immunoglobulin G (IgG) deposition in the kidney. Both mycophenolate and rapamycin, especially the latter, decreased glomerular mTOR Ser2448 phosphorylation. Renal histology in untreated mice showed mesangial proliferation and progressive glomerulosclerosis with tubular atrophy, and increased expression of transforming growth factor β1 (TGF-β1), monocyte chemoattractant protein-1 (MCP-1), α-smooth muscle actin (α-SMA), fibronectin (FN) and collagen. Both mycophenolate and rapamycin ameliorated the histopathological changes. Results from in vitro experiments showed that both mycophenolate and rapamycin decreased mesangial cell proliferation and their binding with anti-dsDNA antibodies. Mycophenolate and rapamycin also down-regulated mTOR and extracellular signal-regulated kinase (ERK) phosphorylation and inhibited fibrotic responses in mesangial cells that were induced by anti-dsDNA antibodies or TGF-β1. Our findings suggest that, in addition to immunosuppression, mycophenolate and rapamycin may reduce fibrosis in LN, which has important implications in preventing CKD in patients with LN.

Rationale: Dysregulated microRNA (miRNA) expressions in cancer can contribute to chemoresistance. This study aims to identify miRNAs that are associated with fluorouracil (5-FU) chemoresistance in esophageal squamous cell carcinoma (ESCC). The potential of miR-29c as a novel diagnostic, prognostic and treatment-predictive marker in ESCC, and its mechanisms and therapeutic implication in overcoming 5-FU chemoresistance were explored. Methods: The miRNA profiles of an ESCC cell model with acquired chemoresistance to 5-FU were analyzed using a Taqman miRNA microarray to identify novel miRNAs associated with 5-FU chemoresistance. Quantitative real-time PCR was used to determine miR-29c expression in tissue and serum samples of patients. Bioinformatics, gain- and loss-of-function experiments, and luciferase reporter assay were performed to validate F-box only protein 31 (FBXO31) as a direct target of miR-29c, and to identify potential transcription factor binding events that control miR-29c expression. The potential of systemic miR-29c oligonucleotide-based therapy in overcoming 5-FU chemoresistance was evaluated in tumor xenograft model. Results: MiR-29c, under the regulatory control of STAT5A, was frequently downregulated in tumor and serum samples of patients with ESCC, and the expression level was correlated with overall survival. Functional studies showed that miR-29c could override 5-FU chemoresistance in vitro and in vivo by directly interacting with the 3'UTR of FBXO31, leading to repression of FBXO31 expression and downstream activation of p38 MAPK. Systemically administered miR-29c dramatically improved response of 5-FU chemoresistant ESCC xenografts in vivo. Conclusions: MiR-29c modulates chemoresistance by interacting with FBXO31, and is a promising non-invasive biomarker and therapeutic target in ESCC.

ObjectivesIn patients with systemic lupus erythematosus (SLE), immune tolerance breakdown leads to autoantibody production and immune-complex glomerulonephritis. This study aimed to identify pathogenic plasma cells (PC) in the development of lupus nephritis.MethodsPC subsets in peripheral blood and renal tissue of patients with SLE and lupus mice were examined by flow cytometry and confocal microscopy, respectively. Sorting-purified PCs from lupus mice were adoptively transferred into Rag2-deficient recipients, in which immune-complex deposition and renal pathology were investigated. In culture, PCs from lupus mice and patients with SLE were treated with a TLR4 inhibitor and examined for autoantibody secretion by enzyme-linked immunospot assay (ELISPOT). Moreover, lupus mice were treated with a TLR4 inhibitor, followed by the assessment of serum autoantibody levels and glomerulonephritis activity.ResultsThe frequencies of TLR4+CXCR4+ PCs in peripheral blood and renal tissue were found significantly increased with the potent production of anti-dsDNA IgG, which were associated with severe renal damages in patients with SLE and mice with experimental lupus. Adoptive transfer of TLR4+CXCR4+ PCs from lupus mice led to autoantibody production and glomerulonephritis development in Rag2-deficient recipients. In culture, TLR4+CXCR4+ PCs from both lupus mice and patients with SLE showed markedly reduced anti-dsDNA IgG secretion on TLR4 blockade. Moreover, in vivo treatment with TLR4 inhibitor significantly attenuated autoantibody production and renal damages in lupus mice.ConclusionsThese findings demonstrate a pathogenic role of TLR4+CXCR4+ PCs in the development of lupus nephritis and may provide new therapeutic strategies for the treatment of SLE.

Objectives To evaluate the use of shear wave elastography in assessment of kidney allograft tubulointerstitial fibrosis. Methods Shear wave elastography assessment was carried out by two independent operators in kidney transplant recipients who underwent allograft biopsy for clinical indications (i.e. rising creatinine >15% or proteinuria >1 g/day). Allograft biopsies were interpreted by the same pathologist according to the 2013 Banff Classification. Results A total of 40 elastography scans were carried out (median creatinine 172.5 μmol/L [interquartile range 133.8-281.8 μmol/L]). Median tissue stiffness at the cortex (22.6 kPa [interquartile range 18.8-25.7 kPa] vs 22.3 kPa [interquartile range 19.0-26.5 kPa], P = 0.70) and medulla (15.0 kPa [interquartile range 13.7-18.0 kPa] vs 15.6 kPa [interquartile range 14.4-18.2 kPa]) showed no significant differences between the two observers. Interobserver agreement was satisfactory (intraclass correlation coefficient of the cortex 0.84, 95% CI 0.70-0.92 and intraclass correlation coefficient of the medulla 0.88, 95% CI 0.78-0.94). The areas under the receiver operating characteristic curves for detection of tubulointerstitial fibrosis were estimated to be 0.75 (95% CI 0.61-0.89), 0.85 (95% CI 0.75-0.95) and 0.65 (95% CI 0.53-0.78) for cortical, medullary tissue stiffness and serum creatinine, respectively. Conclusions Shear wave elastography can be used as a non-invasive tool to evaluate kidney allograft fibrosis with reasonable interobserver agreement and superior test performance to serum creatinine in detecting early tubulointerstitial fibrosis.

Oesophageal squamous cell carcinoma (ESCC) is one of the most lethal cancers, owing to a high frequency of metastasis. However, little is known about the genomic landscape of metastatic ESCC. To identify the genetic alterations that underlie ESCC metastasis, whole-exome sequencing was performed for 41 primary tumours and 15 lymph nodes (LNs) with metastatic ESCCs. Eleven cases included matched primary tumours, synchronous LN metastases, and non-neoplastic mucosa. Approximately 50-76% of the mutations identified in primary tumours appeared in the synchronous LN metastases. Metastatic ESCCs harbour frequent mutations of TP53, KMT2D, ZNF750, and IRF5. Importantly, ZNF750 was recurrently mutated in metastatic ESCC. Combined analysis from current and previous genomic ESCC studies indicated more frequent ZNF750 mutation in diagnosed cases with LN metastasis than in those without metastasis (14% versus 3.4%, n = 629, P = 1.78 × 10-5 ). The Cancer Genome Atlas data further showed that ZNF750 genetic alterations were associated with early disease relapse. Previous ESCC studies have demonstrated that ZNF750 knockdown strongly promotes proliferation, migration, and invasion. Collectively, these results suggest a role for ZNF750 as a metastasis suppressor. TP53 is highly mutated in ESCC, and missense mutations are associated with poor overall survival, independently of pathological stage, suggesting that these missense mutations have important functional impacts on tumour progression, and are thus likely to be gain-of-function (GOF) mutations. Additionally, mutations of epigenetic regulators, including KMT2D, TET2, and KAT2A, and chromosomal 6p22 and 11q23 deletions of histone variants, which are important for nucleosome assembly, were detected in 80% of LN metastases. Our study highlights the important role of critical genetic events including ZNF750 mutations, TP53 putative GOF mutations and nucleosome disorganization caused by genetic lesions seen with ESCC metastasis. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Esophageal cancer is one of the most lethal cancers worldwide with poor survival and limited therapeutic options. The discovery of microRNAs created a new milestone in cancer research. miR-377 is located in chromosome region 14q32, which is frequently deleted in esophageal squamous cell carcinoma (ESCC), but the biological functions, clinical significance and therapeutic implication of miR-377 in ESCC are largely unknown. In this study, we found that miR-377 expression was significantly downregulated in tumor tissue and serum of patients with ESCC. Both tumor tissue and serum miR-377 expression levels were positively correlated with patient survival. Higher serum miR-377 expression was inversely associated with pathologic tumor stage, distant metastasis, residual tumor status and chemoradiotherapy resistance. The roles of miR-377 in suppressing tumor initiation and progression, and the underlying molecular mechanisms were investigated. Results of in vitro and in vivo experiments showed that miR-377 overexpression inhibited the initiation, growth and angiogenesis of ESCC tumors as well as metastatic colonization of ESCC cells, whereas silencing of miR-377 had opposite effects. Mechanistically, miR-377 regulated CD133 and VEGF by directly binding to their 3′ untranslated region. Moreover, systemic delivery of formulated miR-377 mimic not only suppressed tumor growth in nude mice but also blocked tumor angiogenesis and metastasis of ESCC cells to the lungs without overt toxicity to mice. Collectively, our study established that miR-377 plays a functional and significant role in suppressing tumor initiation and progression, and may represent a promising non-invasive diagnostic and prognostic biomarker and therapeutic strategy for patients with ESCC.

// Bin Li 1 , Cian M. McCrudden 2 , Hiu Fung Yuen 3 , Xinping Xi 1 , Peng Lyu 1 , Kwok Wah Chan 4 , Shu Dong Zhang 5 , Hang Fai Kwok 1 1 Faculty of Health Sciences, University of Macau, Avenida de Universidade, Taipa, Macau SAR 2 School of Pharmacy, Queen’s University Belfast, Belfast, United Kingdom 3 Institute of Molecular and Cell Biology, A*STAR, Singapore 4 Department of Pathology, University of Hong Kong, Hong Kong 5 Northern Ireland Centre for Stratified Medicine, Biomedical Sciences Research Institute, University of Ulster, Londonderry, United Kingdom Correspondence to: Hang Fai Kwok, email: hfkwok@umac.mo Keywords: brain tumor, CD133, HOX, LIM2, stem cell Received: April 13, 2016 Accepted: December 26, 2016 Published: December 31, 2016 ABSTRACT CD133 has been shown to be an important stem cell factor that promotes glioma progression. However, the mechanism for CD133-mediated glioma progression has yet to be fully elucidated. In this study, we found that CD133 mRNA expression was a prognostic marker in three independent glioma patient cohorts, corroborating a putative role for CD133 in glioma progression. Importantly, we found that CD133 expression in glioma was highly correlated with the expression of HOX gene stem cell factors (HOXA5, HOXA7, HOXA10, HOXC4 and HOXC6). The expression of these HOX genes individually was significantly associated with survival. Interestingly, the prognostic significance of CD133 was dependent on the expression level of HOX genes, and vice versa. CD133 ( p = 0.021) and HOXA7 ( p = 0.001) were independent prognostic markers when the three glioma patient cohorts were combined ( n = 231). Our results suggest that HOX genes may play a more important role in progression of glioma when CD133 expression is low. Furthermore, we showed that low-level expression of LIM2 in CD133-high glioma was associated with poorer survival, suggesting that LIM2 could be a therapeutic target for glioma expressing a high level of CD133. Connectivity mapping identified vinblastine and vincristine as agents that could reverse the CD133/HOX genes/LIM2-signature, and we confirmed this by in vitro analysis in glioma cell lines, demonstrating that CD133 and HOX genes were co-expressed and could be downregulated by vincristine. In conclusion, our data show that CD133 and HOX genes are important prognostic markers in glioma and shed light on possible treatment strategies for glioma expressing a high level of CD133.

5‐Fluorouracil (5‐FU) is a chemotherapeutic agent commonly used to treat esophageal squamous cell carcinoma (ESCC), but acquisition of chemoresistance frequently occurs and the underlying mechanisms are not fully understood. We found that microRNA (miR)‐338‐5p was underexpressed in ESCC cells with acquired 5‐FU chemoresistance. Forced expression of miR‐338‐5p in these cells resulted in downregulation of Id‐1, and restoration of both in vitro and in vivo sensitivity to 5‐FU treatment. The effects were abolished by reexpression of Id‐1. In contrast, miR‐338‐5p knockdown induced 5‐FU resistance in chemosensitive esophageal cell lines, and knockdown of both miR‐338‐5p and Id‐1 resensitized the cells to 5‐FU. In addition, miR‐338‐5p had suppressive effects on migration and invasion of ESCC cells. Luciferase reporter assay confirmed a direct interaction between miR‐338‐5p and the 3′‐UTR of Id‐1. We also found that miR‐338‐5p was significantly downregulated in tumor tissue and serum samples of patients with ESCC. Notably, low serum miR‐338‐5p expression level was associated with poorer survival and poor response to 5‐FU/cisplatin‐based neoadjuvant chemoradiotherapy. In summary, we found that miR‐338‐5p can modulate 5‐FU chemoresistance and inhibit invasion‐related functions in ESCC by negatively regulating Id‐1, and that serum miR‐338‐5p could be a novel noninvasive prognostic and predictive biomarker in ESCC., We found that microRNA (miR)‐338‐5p was underexpressed in esophageal squamous cell carcinoma cells with acquired 5‐fluorouracil (5‐FU) chemoresistance, and that reexpression of miR‐338‐5p could resensitize them to 5‐FU treatment through targeting Id‐1. MicroRNA‐338‐5p was significantly downregulated in tumor tissue and serum of patients with esophageal squamous cell carcinoma. Low serum miR‐338‐5p was predictive of poor response to 5‐FU/cisplatin‐based neoadjuvant chemoradiotherapy.

Oesophageal squamous cell carcinoma (ESCC) is the most common histological subtype of oesophageal cancer. The disease is particularly prevalent in southern China. The incidence of the disease is on the rise and its overall survival rate remains dismal. Identification and characterization of better molecular markers for early detection and therapeutic targeting are urgently needed. Here, we report levels of transmembrane and soluble neuropilin-2 (NRP2) to be significantly up-regulated in ESCC, and to correlate positively with advanced tumour stage, lymph node metastasis, less favourable R category and worse overall patient survival. NRP2 up-regulation in ESCC was in part a result of gene amplification at chromosome 2q. NRP2 overexpression promoted clonogenicity, angiogenesis and metastasis in ESCC in vitro, while NRP2 silencing by lentiviral knockdown or neutralizing antibody resulted in a contrary effect. This observation was extended in vivo in animal models of subcutaneous tumourigenicity and tail vein metastasis. Mechanistically, overexpression of NRP2 induced expression of ERK MAP kinase and the transcription factor ETV4, leading to enhanced MMP-2 and MMP-9 activity and, as a consequence, suppression of E-cadherin. In summary, NRP2 promotes tumourigenesis and metastasis in ESCC through deregulation of ERK-MAPK-ETV4-MMP-E-cadherin signalling. NRP2 represents a potential diagnostic or prognostic biomarker and therapeutic target for ESCC. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

// Jian Li 1, 2, * , Bao-Zhu Zhang 1, * , Yan-Ru Qin 3 , Jiong Bi 4 , Hai-Bo Liu 1, 5 , Yan Li 1 , Mu-Yan Cai 1, 6 , Stephanie Ma 7 , Kwok Wah Chan 8 , Dan Xie 1 , Xin-Yuan Guan 1, 9 1 State Key Laboratory of Oncology in Southern China, Sun Yat-Sen University Cancer Center, Guangzhou, China 2 Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China 3 Department of Clinical Oncology, The First Affiliated Hospital, Zhengzhou University, Zhengzhou, China 4 Department of Surgery, The First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, China 5 Key Laboratory for Major Obstetric Diseases of Guangdong Province, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou Medical University, Guangzhou, China 6 Department of Pathology, Sun Yat-Sen University Cancer Center, Sun Yat-Sen University, Guangzhou, China 7 Department of Anatomy, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China 8 Department of Pathology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China 9 Department of Clinical Oncology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China * These authors contributed equally to this work Correspondence to: Xin-Yuan Guan, e-mail: xyguan@hkucc.hku.hk Keywords: ESCC, IL-13, macrophage, prognosis, CD68 Received: August 14, 2015 Accepted: December 01, 2015 Published: January 12, 2016 ABSTRACT Purpose: Oncology immunity was reported to play a key role in cancer development and progression, so we investigated the prediction role of several immune markers in esophageal squamous cell carcinoma (ESCC) patients after operation in this study. Patients and Methods: 66 primary ESCC tumor tissues and four sets of tissue microarrays including 705 primary ESCC tumor tissues from four centers were collected and analyzed. Expressions of several immune markers in ESCC tumor tissue were detected with immunohistochemistry staining. Their distribution densities were analyzed with InForm ™ 2.0.1 software. All statistic analyses were performed with SPSS16.0 and Stata version 10.0. Results: Survival analyses assessed by Kaplan-Meier plots and log-rank tests demonstrated that densities of CD68 and interleukin 13 (IL-13) in tumor stroma were positively correlated with the overall survival of ESCC patients after operation ( p < 0.01 for CD68, p < 0.001 for IL-13). Further, a model based on tumor stroma densities of CD68 and IL-13 was constructed and it could significantly classify patients with poor or good prognosis. This model could further identify high-risk group and low-risk group at the same Tumor lymph Nodes Metastases (TNM) stage. Lastly, a more accuracy model based on TNM stage, densities of CD68 and IL-13 was constructed to predict the prognosis of ESCC patient after operation. Conclusion: Combining the TNM staging system and densities of CD68 and IL-13 could substantially improve the prognosis prediction accuracy of ESCC patient after operation, which might be an excellent tool for selecting patients for individualized therapy in future.

Emerging evidence suggests that activation of adenosine monophosphate-activated protein kinase (AMPK) may suppress cancer growth. Identification of novel AMPK activators is therefore crucial to exploit AMPK as a potential target for cancer prevention and treatment.We determined the expression status and role of AMPK in esophageal squamous cell carcinoma (ESCC) and investigated whether silibinin, a nontoxic natural product, could activate AMPK to inhibit ESCC development.Our results from 49 pairs of human ESCC and normal tissues showed that AMPK was constitutively inactive in the majority (69.4%) of ESCC. We found that silibinin induced apoptosis, and inhibited ESCC cell proliferation in vitro and tumorigenicity in vivo without any adverse effects. Silibinin also markedly suppressed the invasive potential of ESCC cells in vitro and their ability to form lung metastasis in nude mice. The anticancer effects of silibinin were abrogated by the presence of compound C or shRNA against AMPK. More importantly, silibinin enhanced the sensitivity of ESCC cells and tumors to the chemotherapeutic drugs, 5-fluorouracil and cisplatin.This preclinical study supports that AMPK is a valid therapeutic target and suggests that silibinin may be a potentially useful therapeutic agent and chemosensitizer for esophageal cancer.

Esophageal squamous cell carcinoma (ESCC) is the most predominantly occurring type of esophageal cancer worldwide. Locally advanced ESCC patients are treated by neoadjuvant chemoradiation for tumor downstaging prior to tumor resection. Patients receiving this treatment have an increased expectation of cure via the following tumor resection and have better survival outcomes. However, not all patients respond well to chemoradiation and poor responders suffer from treatment-associated toxicity and complications without benefits. No method is currently available to predict patient chemoradiation response and to exclude poor responders from ineffective treatment. To address this clinical limitation, the present study aimed to identify non-invasive biomarkers for predicting patient chemoradiation response. Due to the features of microRNA (miRNA) in cancer diagnosis, prognosis and treatment response prediction, serum miRNA arrays were performed to identify potential miRNA(s) that may be used for chemoradiation response prediction in ESCC. Using an miRNA array to compare pre-treatment serum sample pools from 10 good responders and 10 poor responders, the present study identified miR-193b, miR-942 and miR-629* as candidate miRNAs for predicting chemoradiation response. Subsequent validation using reverse transcription-quantitative polymerase chain reaction confirmed that miR-193b, however not miR-942 and miR-629*, were significantly increased in sera from 24 good responders, compared with 23 poor responders. Further analyses using the receiver operating characteristic curve revealed a strong predictive power of serum miR-193b on discriminating good responders from poor responders to chemoradiation. In addition, a high serum level of miR-193b was significantly associated with better survival outcomes. Therefore, serum miR-193b may be considered a promising biomarker for predicting chemoradiation response and post-therapy survival of ESCC patients.

JC virus (JCV)-associated nephropathy has been increasingly recognized as a cause of allograft dysfunction with graft loss in renal transplant recipients. Like many other opportunistic viral infections in transplant recipients, there are currently limited therapeutic options for this condition. Fusidic acid has previously been reported to exhibit antiviral activity against JCV in in vitro assays. We report the first in vivo study to document the rapid reduction of JC viruria and stabilization of allograft function by oral fusidic acid (fusidate sodium) in a deceased donor renal transplant recipient with JCV-associated nephropathy and acute allograft dysfunction which did not improve initially to surgical relief of hydronephrosis and reduction of immunosuppressants. Rapid reduction of JC viruria detected by quantitative PCR and stabilization of renal function were observed. Fusidic acid has several practical advantages in this clinical setting, including a low EC50 against JCV, high plasma C max, long half-life, availability of both oral and intravenous formulations, excellent oral bioavailability, good patient tolerability, and lack of serious drug interactions with other drugs taken by renal transplant recipients. Further mechanistic and clinical studies are necessary to evaluate this treatment option for JCV-associated nephropathy.

Immunotherapy is a promising strategy for the treatment of various types of cancer. An antibody that targets programmed death ligand-1 (PD-L1) pathway has been shown to be active towards various types of cancer, including melanoma and lung cancer. MPDL3280A, an anti-PD-L1 antibody, has shown clear clinical activity in PD-L1-overexpressing bladder cancer with an objective response rate of 40-50%, resulting in a breakthrough therapy designation granted by FDA. These events pronounce the importance of targeting the PD-L1 pathway in the treatment of bladder cancer. In the present study, we investigated the prognostic significance of the expression of three genes in the PD-L1 pathway, including PD-L1, B7.1 and PD-1, in three independent bladder cancer datasets in the Gene Expression Omnibus database. PD-L1, B7.1 and PD-1 were significantly associated with clinicopathological parameters indicative of a more aggressive phenotype of bladder cancer, such as a more advanced stage and a higher tumor grade. In addition, a high level expression of PD-L1 was associated with reduced patient survival. Of note, the combination of PD-L1 and B7.1 expression, but not other combinations of the three genes, were also able to predict patient survival. Our findings support the development of anti-PD-L1, which blocks PD-L1-PD-1 and B7.1-PD-L1 interactions, in treatment of bladder cancer. The observations were consistent in the three independent bladder cancer datasets consisting of a total of 695 human bladder specimens. The datasets were then assessed and it was found that the expression levels of the chemokine CC-motif ligand (CCL), CCL3, CCL8 and CCL18, were correlated with the PD-L1 expression level, while ADAMTS13 was differentially expressed in patients with a different survival status (alive or deceased). Additional investigations are required to elucidate the role of these genes in the PD-L1-mediated immune system suppression and bladder cancer progression. In conclusion, findings of this study suggested that PD-L1 is an important prognostic marker and a therapeutic target for bladder cancer.

The level of anti-dsDNA antibodies correlates with disease activity in lupus nephritis, but their role in pathogenic mechanisms remains to be defined. We investigated the effect of anti-dsDNA antibodies isolated from lupus nephritis patients on fibronectin synthesis and downstream fibrogenesis in proximal renal tubular epithelial cells (PTEC). Kidney biopsies were obtained from patients with active severe proliferative lupus nephritis. In vitro studies with cultured PTEC were performed to investigate the effect of human polyclonal IgG anti-dsDNA antibodies and mycophenolic acid (MPA). The role of IL-6, IL-8, MCP-1, TNF-α, TGF-β1, and MAPK and PKC signaling pathways on soluble and cell-associated fibronectin synthesis was investigated using neutralizing antibodies or specific inhibitors. The effect of exogenous endotoxin-free soluble fibronectin on downstream fibrotic processes was also examined. Fibronectin expression was markedly increased in the tubulo-interstitium of lupus nephritis renal biopsies and it co-localized with IgG deposition. Anti-dsDNA antibodies significantly increased both secreted and cell-associated fibronectin, through prior activation of ERK, p38 MAPK, JNK, PKC-α and PKC-βII. There was downstream induction of IL-6, IL-8, MCP-1, TNF-α and TGF-β1. MPA inhibited the induction of inflammatory and fibrotic processes by anti-dsDNA antibody. Exogenous soluble fibronectin induced TGF-β1 secretion and type I collagen synthesis in PTEC in a dose-dependent manner. Our data demonstrate that anti-dsDNA antibody contributes to tubulo-interstitial fibrosis in lupus nephritis through its action on PTEC. Anti-dsDNA antibody induces both cell-associated and soluble fibronectin secretion in PTEC, the former adds to extracellular matrix deposition while the latter amplifies the fibrotic process through induction of TGF-β1 and collagen type I. The pro-fibrotic effects of anti-dsDNA antibody are ameliorated by MPA.

// Wen Wen Xu 1, 2 , Bin Li 1, 2, 3 , Alfred KY Lam 4 , Sai Wah Tsao 1, 3 , Simon YK Law 3, 5 , Kwok Wah Chan 2, 3, 6 , Qiu Ju Yuan 1 , Annie LM Cheung 1, 2, 3 1 Department of Anatomy, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong SAR, China 2 The University of Hong Kong-Shenzhen Institute of Research and Innovation (HKU-SIRI), China 3 Centre for Cancer Research, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong SAR, China 4 Department of Pathology, Griffith Medical School and Griffith Health Institute, Gold Coast Campus, Gold Coast, QLD 4222, Australia 5 Department of Surgery, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong SAR, China 6 Department of Pathology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong SAR, China Correspondence to: Annie LM Cheung, e-mail: lmcheung@hku.hk Keywords: Tumor angiogenesis, Bone marrow-derived cells, Tumor microenvironment, VEGF receptors, Antibody therapy Received: September 18, 2014 Accepted: November 19, 2014 Published: December 17, 2014 ABSTRACT Increasing appreciation of tumor heterogeneity and the tumor-host interaction has stimulated interest in developing novel therapies that target both tumor cells and tumor microenvironment. Bone marrow derived cells (BMDCs) constitute important components of the tumor microenvironment. In this study, we aim to investigate the significance of VEGFR1- and VEGFR2-expressing non-tumor cells, including BMDCs, in esophageal cancer (EC) progression and in VEGFR1/VEGFR2-targeted therapies. Here we report that VEGFR1 or VEGFR2 blockade can significantly attenuate VEGF-induced Src and Erk signaling, as well as the proliferation and migration of VEGFR1 + and VEGFR2 + bone marrow cells and their pro-invasive effect on cancer cells. Importantly, our in vivo data show for the first time that systemic blockade of VEGFR1 + or VEGFR2 + non-tumor cells with neutralizing antibodies is sufficient to significantly suppress esophageal tumor growth, angiogenesis and metastasis in mice. Moreover, our tissue microarray study of human EC clinical specimens showed the clinicopathological significance of VEGFR1 and VEGFR2 in EC, which suggest that anti-VEGFR1/VEGFR2 therapies may be particularly beneficial for patients with aggressive EC. In conclusion, this study demonstrates the important contributions of VEGFR1 + and VEGFR2 + non-tumor cells in esophageal cancer progression, and substantiates the validity of these receptors as therapeutic targets for this deadly disease.

Local interactions between cancer cells and stroma can produce systemic effects on distant organs to govern cancer progression. Here we show that IGF2 secreted by inhibitor of differentiation (Id1)-overexpressing oesophageal cancer cells instigates VEGFR1-positive bone marrow cells in the tumour macroenvironment to form pre-metastatic niches at distant sites by increasing VEGF secretion from cancer-associated fibroblasts. Cancer cells are then attracted to the metastatic site via the CXCL5/CXCR2 axis. Bone marrow cells transplanted from nude mice bearing Id1-overexpressing oesophageal tumours enhance tumour growth and metastasis in recipient mice, whereas systemic administration of VEGFR1 antibody abrogates these effects. Mechanistically, IGF2 regulates VEGF in fibroblasts via miR-29c in a p53-dependent manner. Analysis of patient serum samples showed that concurrent elevation of IGF2 and VEGF levels may serve as a prognostic biomarker for oesophageal cancer. These findings suggest that the Id1/IGF2/VEGF/VEGFR1 cascade plays a critical role in tumour-driven pathophysiological processes underlying cancer progression.

Purpose: To investigate the autocrine/endocrine role of Id1-induced insulin-like growth factor-II (IGF-II) in esophageal cancer, and evaluate the potential of IGF-II- and IGF-type I receptor (IGF-IR)-targeted therapies. Experimental Design: Antibody array-based screening was used to identify differentially secreted growth factors from Id1-overexpressing esophageal cancer cells. In vitro and in vivo assays were performed to confirm the induction of IGF-II by Id1, and to study the autocrine and endocrine effects of IGF-II in promoting esophageal cancer progression. Human esophageal cancer tissue microarray was analyzed for overexpression of IGF-II and its correlation with that of Id1 and phosphorylated AKT (p-AKT). The efficacy of intratumorally injected IGF-II antibody and intraperitoneally injected cixutumumab (fully human monoclonal IGF-IR antibody) was evaluated using in vivo tumor xenograft and experimental metastasis models. Results: Id1 overexpression induced IGF-II secretion, which promoted cancer cell proliferation, survival, and invasion by activating AKT in an autocrine manner. Overexpression of IGF-II was found in 21 of 35 (60%) esophageal cancer tissues and was associated with upregulation of Id1 and p-AKT. IGF-II secreted by Id1-overexpressing esophageal cancer xenograft could instigate the growth of distant esophageal tumors, as well as promote metastasis of circulating cancer cells. Targeting IGF-II and IGF-IR had significant suppressive effects on tumor growth and metastasis in mice. Cixutumumab treatment enhanced the chemosensitivity of tumor xenografts to fluorouracil and cisplatin. Conclusions: The Id1–IGF-II–IGF-IR–AKT signaling cascade plays an important role in esophageal cancer progression. Blockade of IGF-II/IGF-IR signaling has therapeutic potential in the management of esophageal cancer. Clin Cancer Res; 20(10); 2651–62. ©2014 AACR.

Non-CG methylation has been associated with stemness regulation in embryonic stem cells. By comparing differentially expressed genes affected by non-CG methylation between tumour and corresponding non-tumour tissues in oesophageal squamous cell carcinoma (OSCC), we find that Integrin α7 (ITGA7) is characterized as a potential cancer stem cell (CSC) marker. Clinical data show that a high frequency of ITGA7+ cells in OSCC tissues is significantly associated with poor differentiation, lymph node metastasis and worse prognosis. Functional studies demonstrate that both sorted ITGA7+ cells and ITGA7 overexpressing cells display enhanced stemness features, including elevated expression of stemness-associated genes and epithelial–mesenchymal transition features, as well as increased abilities to self-renew, differentiate and resist chemotherapy. Mechanistic studies find that ITGA7 regulates CSC properties through the activation of the FAK-mediated signalling pathways. As knockdown of ITGA7 can effectively reduce the stemness of OSCC cells, ITGA7 could be a potential therapeutic target in OSCC treatment., There is still no consensus on tumour type-specific cancer stem cell markers. Here, the authors demonstrate that ITGA7 is a potential functional marker of oesophageal cancer stem cells involved in the resistance to chemotherapy and metastasis through activation of FAK-mediated signalling.

Prostate cancer is one of the most common cancers in men worldwide, and the number of diagnosed patients has dramatically increased in recent years. Currently, the clinical parameters used to diagnose prostate cancer, such as Gleason score, pathological tumor staging, and prostate-specific antigen (PSA) expression level, are considered insufficient to inform recommendation to guide clinical practice. Thus, identification of a novel biomarker is necessary. TWIST is one of the well-studied targets and is correlated with cancer invasion and metastasis in several human cancers. We have investigated two largest prostate cancer patient cohorts available in GEO database and found that TWIST expression is positive correlated with Gleason score and associated with poorer survival. By using a prostate cancer cohort and a prostate cancer cell line dataset, we have identified three potential downstream targets of TWIST, PPM1A, SRP72 and TBCB. TWIST's prognostic capacity is lost when the gene is mutated. Further investigation in the prostate cancer cohort revealed that gene expression of SERPINA, STX7, PDIA2, FMP5, GP1BB, VGLL4, KCNMA1, SHMT2, SAA4 and DIDO1 influence the prognostic significance of TWIST and vice versa. Importantly, eight out of these ten genes are prognostic indicator by itself. In conclusion, our study has further confirmed that TWIST is a prognostic marker in prostate cancer, identified its potential downstream targets and genes that could possibly give additional prognostic value to predict TWIST-mediated prostate cancer progression.

Esophageal squamous cell carcinoma (ESCC), the major histologic subtype of esophageal cancer, is a devastating disease characterized by distinctly high incidences and mortality rates. However, there remains limited understanding of molecular events leading to development and progression of the disease, which are of paramount importance to defining biomarkers for diagnosis, prognosis, and personalized treatment. By high-throughout transcriptome sequence profiling of nontumor and ESCC clinical samples, we identified a subset of significantly differentially expressed genes involved in integrin signaling. The Rab25 gene implicated in endocytic recycling of integrins was the only gene in this group significantly downregulated, and its downregulation was confirmed as a frequent event in a second larger cohort of ESCC tumor specimens by quantitative real-time PCR and immunohistochemical analyses. Reduced expression of Rab25 correlated with decreased overall survival and was also documented in ESCC cell lines compared with pooled normal tissues. Demethylation treatment and bisulfite genomic sequencing analyses revealed that downregulation of Rab25 expression in both ESCC cell lines and clinical samples was associated with promoter hypermethylation. Functional studies using lentiviral-based overexpression and suppression systems lent direct support of Rab25 to function as an important tumor suppressor with both anti-invasive and -angiogenic abilities, through a deregulated FAK–Raf–MEK1/2–ERK signaling pathway. Further characterization of Rab25 may provide a prognostic biomarker for ESCC outcome prediction and a novel therapeutic target in ESCC treatment. Cancer Res; 72(22); 6024–35. ©2012 AACR.

Annexin II on mesangial cell surface mediates the binding of anti-dsDNA antibodies and consequent downstream inflammatory and fibrotic processes. We investigated the clinical relevance of circulating annexin II-binding immunoglobulins (Igs) in patients with severe proliferative lupus nephritis, and renal annexin II expression in relation to progression of nephritis in New Zealand Black and White F1 mice (NZBWF1/J) mice. Annexin II-binding Igs in serum were measured by ELISA. Ultrastructural localization of annexin II was determined by electron microscopy. Seropositivity rates for annexin II-binding IgG and IgM in patients with active lupus nephritis were significantly higher compared with controls (8.9%, 1.3% and 0.9% for annexin II-binding IgG and 11.1%, 4.0% and 1.9% for annexin II-binding IgM for patients with active lupus nephritis, patients with non-lupus renal disease and healthy subjects respectively). In lupus patients, annexin II-binding IgM level was higher at disease flare compared with remission. Annexin II-binding IgG and IgM levels were associated with that of anti-dsDNA and disease activity. Annexin II-binding IgG and IgM levels correlated with histological activity index in lupus nephritis biopsy samples. In NZBWF1/J mice, serum annexin II-binding IgG and IgM levels and glomerular annexin II and p11 expression increased with progression of active nephritis. Annexin II expression was present on mesangial cell surface and in the mesangial matrix, and co-localized with electron-dense deposits along the glomerular basement membrane. Our results show that circulating annexin II-binding IgG and IgM levels are associated with clinical and histological disease activity in proliferative lupus nephritis. The co-localization of annexin II and p11 expression with immune deposition in the kidney suggests pathogenic relevance.

Mutation of FAM134B (Family with Sequence Similarity 134, Member B) leading to loss of function of its encoded Golgi protein and has been reported induce apoptosis in neurological disorders. FAM134B mutation is still unexplored in cancer. Herein, we studied the DNA copy number variation and novel mutation sites of FAM134B in a large cohort of freshly collected oesophageal squamous cell carcinoma (ESCC) tissue samples. In ESCC tissues, 37% (38/102) showed increased FAM134B DNA copies whereas 35% (36/102) showed loss of FAM134B copies relative to matched non-cancer tissues. Novel mutations were detected in exons 4, 5, 7, 9 as well as introns 2, 4-8 of FAM134B via HRM (High-Resolution Melt) and Sanger sequencing analysis. Overall, thirty-seven FAM134B mutations were noted in which most (31/37) mutations were homozygous. FAM134B mutations were detected in all the cases with metastatic ESCC in the lymph node tested and in 14% (8/57) of the primary ESCC. Genetic alteration of FAM134B is a frequent event in the progression of ESCCs. These findings imply that mutation might be the major driving source of FAM134B genetic modulation in ESCCs.

// Kai Yau Wong 1 , Jing Liu 1, * and Kwok Wah Chan 1 1 Department of Pathology, The University of Hong Kong, Hong Kong * Co-first author Correspondence to: Kwok Wah Chan, email: kwchan@pathology.hku.hk Keywords: KIF7, tumor suppressor gene, prostate cancer, LKB1 Received: July 04, 2016 Accepted: January 10, 2017 Published: April 26, 2017 ABSTRACT This study investigated kinesin family member 7 (KIF7) expression and function in prostate cancer (PCa). Our results showed that KIF7 was significantly downregulated in PCa, compared with normal, benign prostatic hyperplasia and prostate intraepithelial neoplasia tissues, partially through promoter hypermethylation. We further investigated the effects of KIF7 coiled coil (CC) domain and motor domain (MD) on PCa development in vitro and in vivo . Our results showed that KIF7-CC but not KIF7-MD significantly attenuated proliferation and colony formation, impeded migration and invasion, induced apoptosis and sensitized PCa cells to paclitaxel. Further analysis revealed that KIF7-CC enhanced LKB1 expression and phosphorylation at Ser 428 , which induced PTEN phosphorylation at Ser 380 /Thr 382/383 and consequently blocked AKT phosphorylation at Ser 473 . Downregulation of LKB1 significantly attenuated the suppressive effects of KIF7-CC on cell proliferation, colony formation and AKT phosphorylation. Furthermore, our in vivo studies showed that KIF7-CC reduced prostate tumorigenesis in cell-derived xenografts. Downregulation of LKB1 abrogated the anti-tumor effects of KIF7-CC in these xenografts. Taken together, these findings provide the first evidence to support the role of KIF7 as a negative regulator that inhibits PCa development partially through LKB1-mediated AKT inhibition.

Wnt/β-catenin signaling is activated in CD133 liver cancer stem cells (CSCs), a subset of cells known to be a root of tumor recurrence and therapy resistance in hepatocellular carcinoma (HCC). However, the regulatory mechanism of this pathway in CSCs remains unclear. Here, we show that human microRNA (miRNA), miR-1246, promotes cancer stemness, including self-renewal, drug resistance, tumorigencity, and metastasis, by activation of the Wnt/β-catenin pathway through suppressing the expression of AXIN2 and glycogen synthase kinase 3β (GSK3β), two key members of the β-catenin destruction complex. Clinically, high endogenous and circulating miR-1246 was identified in HCC clinical samples and correlated with a worse prognosis. Further functional analysis identified octamer 4 (Oct4) to be the direct upstream regulator of miR-1246, which cooperatively drive β-catenin activation in liver CSCs. Conclusion: These findings uncover the noncanonical regulation of Wnt/β-catenin in liver CSCs by the Oct4/miR-1246 signaling axis, and also provide a novel diagnostic marker as well as therapeutic intervention for HCC. (Hepatology 2016;64:2062-2076).

Background Urine from kidney transplant recipient has proven to be a viable source for donor DNA. However, an optimized protocol would be required to determine mis-matched donor HLA specificities in view of the scarcity of DNA obtained in some cases. Methods In this study, fresh early morning urine specimens were obtained from 155 kidney transplant recipients with known donor HLA phenotype. DNA was extracted and typing of HLA-A, B and DRB1 loci by polymerase chain reaction-specific sequence primers was performed using tailor-made condition according to the concentration of extracted DNA. Results HLA typing of DNA extracted from urine revealed both recipient and donor HLA phenotypes, allowing the deduction of the unknown donor HLA and hence the degree of HLA mis-match. By adopting the modified procedures, mis-matched donor HLA phenotypes were successfully deduced in all of 35 tested urine samples at DNA quantities spanning the range of 620–24,000 ng. Conclusions This urine-based method offers a promising and reliable non-invasive means for the identification of mis-matched donor HLA antigens in kidney transplant recipients with unknown donor HLA phenotype or otherwise inadequate donor information.

Significance Host genetics, environmental factors, and EBV infection together contribute to nasopharyngeal carcinoma (NPC) development. A number of critical genetic and epigenetic events contributing to tumor development has been reported. However, the genomic alterations in NPC have not been completely deciphered. We used the whole-exome sequencing approach to study the somatic mutations in NPC, and an APOBEC-mediated mutagenesis signature was revealed. Importantly, multiple loss-of-function mutations in the NF-κB–negative regulators ( NFKBIA , CYLD , and TNFAIP3 ) were discovered in NPC tumors, and we functionally confirmed that the NFKBIA loss-of-function mutations induce damaging effects on the WT proteins. Detection of these mutations emphasizes the critical role of NF-κB signaling in NPC tumorigenesis and provides perspectives for targeting this pathway in NPC treatment.

Studies on the role of Wnt/beta-catenin signaling in different forms of kidney disease have yielded discrepant results. Here, we report the biphasic change of renal beta-catenin expression in mice with overload proteinuria in which beta-catenin was upregulated at the early stage (4 weeks after disease induction) but abrogated at the late phase (8 weeks). Acute albuminuria was observed at 1 week after bovine serum albumin injection, followed by partial remission at 4 weeks that coincided with overexpression of renal tubular beta-catenin. Interestingly, a rebound in albuminuria at 8 weeks was accompanied by downregulated tubular beta-catenin expression and heightened tubular apoptosis. In addition, there was an inverse relationship between Dickkopf-3 (Dkk-3) and renal tubular beta-catenin expression at these time points. In vitro, a similar trend in beta-catenin expression was observed in human kidney-2 (HK-2) cells with acute (upregulation) and prolonged (downregulation) exposure to albumin. Induction of a proapoptotic phenotype by albumin was significantly enhanced by silencing beta-catenin in HK-2 cells. Finally, Dkk-3 expression and secretion was increased after prolonged exposure to albumin, leading to the suppression of intracellular beta-catenin signaling pathway. The effect of Dkk-3 on beta-catenin signaling was confirmed by incubation with exogenous Dkk-3 in HK-2 cells. Taken together, these data suggest that downregulation of tubular beta-catenin signaling induced by Dkk-3 has a detrimental role in chronic proteinuria, partially through the increase in apoptosis., published_or_final_version

Nidogen-2 (NID2) is a key component of the basement membrane that stabilizes the extracellular matrix (ECM) network. The aim of the study is to analyze the functional roles of NID2 in the pathogenesis of nasopharyngeal carcinoma (NPC) and esophageal squamous cell carcinoma (ESCC). We performed genome-wide methylation profiling of NPC and ESCC and validated our findings using the methylation-sensitive high-resolution melting (MS-HRM) assay. Results showed that promoter methylation of NID2 was significantly higher in NPC and ESCC samples than in their adjacent non-cancer counterparts. Consistently, down-regulation of NID2 was observed in the clinical samples and cell lines of both NPC and ESCC. Re-expression of NID2 suppresses clonogenic survival and migration abilities of transduced NPC and ESCC cells. We showed that NID2 significantly inhibits liver metastasis. Mechanistic studies of signaling pathways also confirm that NID2 suppresses the EGFR/Akt and integrin/ FAK/PLCγ metastasis-related pathways. This study provides novel insights into the crucial tumor metastasis suppression roles of NID2 in cancers., published_or_final_version

Emerging evidence has indicated a role of the bone morphogenetic proteins (BMP) in the pathogenesis of certain cancers. The signaling of BMP family members is tightly regulated by their antagonists, including noggin and SOST, which are, in turn, positively regulated by BMP, thereby forming a negative feedback loop. Consequently, the expression of these antagonists should be taken into account in studies on the prognostic significance of BMP. In the present paper, we correlated protein and mRNA expression levels of BMP6, noggin and SOST, alone or in combination, with patient survival in various types of cancer. We found that BMP6 alone was not significantly correlated with esophageal squamous cell carcinoma patient survival. Instead, a high level of inhibitor of differentiation 1, a downstream factor of BMP6, was associated with shorter survival in patients whose tumors stained strongly for BMP6. Knockdown of noggin in esophageal cancer cell line EC109, which expresses BMP6 strongly and SOST weakly, enhanced the non‐adherent growth of the cells. Noggin and SOST expression levels, when analyzed alone, were not significantly correlated with patient survival. However, high BMP6 activity, defined by strong BMP6 expression coupled with weak noggin or SOST expression, was significantly associated with shorter survival in esophageal squamous cell carcinoma patients. We further confirmed that BMP6 activity could be used as a prognostic indicator in prostate, bladder and colorectal cancers, using publicly available data on BMP6, noggin and SOST mRNA expression and patient survival. Our results strongly suggest that BMP6, noggin and SOST could be used in combination as a prognostic indicator in cancer progression. (Cancer Sci 2012; 103: 1145–1154)

Polyoma BK virus (BKV) has recently been identified to cause renal allograft dysfunction, which manifests as polyomavirus-associated nephropathy (PVAN). However, the presence and level of BKV DNA in renal allograft patients with good and stable renal function have remained undetermined.In this prospective study, serum samples were collected from a total of 45 renal allograft recipients with serum creatinine155 μmol/L. In 17 patients, whose duration of transplantation was under 2 years, samples were collected at 3-4-month intervals for up to 2 years after transplantation. BK viral load was quantified using quantitative polymerase chain reaction (Q-PCR).The BK viral load in asymptomatic renal allograft recipients was independent of the duration of transplantation and did not correlate with allograft function. The mean (± SD) level of viremia was 552.80 ± 1931.00 genome copies/mL, with 92.9% of patients having low levels of viremia corresponding to1 × 10(3) copies/mL. In contrast, patients with proven PVAN had levels in the range of 10(6) copies/mL.The prevailing BK viral load in asymptomatic renal allograft patients is quantifiably low. Our findings may guide optimal immunosuppressive modulation in PVAN cases, where judicious manipulation of immunosuppression is required without inciting allograft rejection.

Esophageal cancer is a deadly cancer with esophageal squamous cell carcinoma (ESCC) as the major type. Until now there has been a lack of reliable prognostic markers for this malignancy. This study aims to investigate the clinical correlation between Forkhead box M1 (FoxM1) and patients' parameters in ESCC. Methods: Immunohistochemistry was performed to investigate the expression and localization of FoxM1 in 64 ESCC tissues and 10 nontumor esophageal tissues randomly selected from 64 patients before these data were used for clinical correlations. Results: Cytoplasmic and nuclear expressions of FoxM1 were found in 63 and 16 of the 64 ESCC tissues, respectively. Low cytoplasmic expression of FoxM1 was correlated with early pathological stage in ESCC (P = 0.018), while patients with nuclear FoxM1 were younger in age than those without nuclear expression (P, published_or_final_version, Springer Open Choice, 21 Feb 2012

Suppressive effects of DUSP6 in tumorigenesis and EMT-associated properties were observed. Dual-specificity phosphatase (DUSP6) is a MAP kinase phosphatase (MKP) negatively regulating the activity of ERK, one of the major molecular switches in the MAPK signaling cascade propagating the signaling responses during malignancies. The impact of DUSP6 in EMT and its contribution to tumor dissemination has not yet been characterized. Due to differences in tumor microenvironments affecting cell signaling during cancer progression, DUSP6 may play varying roles in tumor development. We sought to examine the potential role of DUSP6-mediated tumorigenesis and EMT-associated properties in two aerodigestive tract cancers, namely, esophageal squamous cell carcinoma (ESCC) and nasopharyngeal carcinoma (NPC). Significant loss of DUSP6 was observed in 100% of 11 ESCC cell lines and 71% of seven NPC cell lines. DUSP6 expression was down-regulated in 40% of 30 ESCC tumor tissues and 75% of 20 NPC tumor tissues compared to their respective normal counterparts. Suppressive effects of DUSP6 in tumor formation and cancer cell mobility are seen in in vivo tumorigenicity assay and in vitro colony formation, three-dimensional Matrigel culture, cell migration and invasion chamber tests. Notably, overexpression of DUSP6 impairs EMT-associated properties. Furthermore, tissue microarray analysis reveals a clinical association of DUSP6 expression with better patient survival. Taken together, our study provides a novel insight into understanding the functional impact of DUSP6 in tumorigenesis and metastasis of ESCC and NPC.

The association of Matrix metalloproteinase-19 (MMP19) in the development of nasopharyngeal carcinoma (NPC) was identified from differential gene profiling, which showed MMP19 was one of the candidate genes down-regulated in the NPC cell lines. In this study, quantitative RT-PCR and Western blot analysis showed MMP19 was down-regulated in all seven NPC cell lines. By tissue microarray immunohistochemical staining, MMP19 appears down-regulated in 69.7% of primary NPC specimens. Allelic deletion and promoter hypermethylation contribute to MMP19 down-regulation. We also clearly demonstrate that the catalytic activity of MMP19 plays an important role in antitumor and antiangiogenesis activities in comparative studies of the wild-type and the catalytically inactive mutant MMP19. In the in vivo tumorigenicity assay, only the wild-type (WT), but not mutant, MMP19 transfectants suppress tumor formation in nude mice. In the in vitro colony formation assay, WT MMP19 dramatically reduces colony-forming ability of NPC cell lines, when compared to the inactive mutant. In the tube formation assay of human umbilical vein endothelial cells and human microvascular endothelial cells (HMEC-1), secreted WT MMP19, but not mutant MMP19, induces reduction of tube-forming ability in endothelial cells with decreased vascular endothelial growth factor (VEGF) in conditioned media detected by enzyme-linked immunosorbent assay (ELISA). The anti-angiogenic activity of WT MMP19 is correlated with suppression of tumor formation. These results now clearly show that catalytic activity of MMP19 is essential for its tumor suppressive and anti-angiogenic functions in NPC.

Polyomavirus enhancer activator 3 protein (Pea3), also known as ETV4, is a member of the Ets-transcription factor family, which promotes metastatic progression in various types of solid cancer. Pea3-driven epithelial-mesenchymal transition (EMT) has been described in lung and ovarian cancers. The mechanisms of Pea3-induced EMT, however, are largely unknown. Here we show that Pea3 overexpression promotes EMT in human breast epithelial cells through transactivation of Snail (SNAI1), an activator of EMT. Pea3 binds to the human Snail promoter through the two proximal Pea3 binding sites and enhances Snail expression. In addition, knockdown of Pea3 in invasive breast cancer cells results in down-regulation of Snail, partial reversal of EMT, and reduced invasiveness in vitro. Moreover, knockdown of Snail partially rescues the phenotype induced by Pea3 overexpression, suggesting that Snail is one of the mediators bridging Pea3 and EMT, and thereby metastatic progression of the cancer cells. In four breast cancer patient cohorts whose microarray and survival data were obtained from the Gene Expression Omnibus database, Pea3 and Snail expression are significantly correlated with each other and with overall survival of breast cancer patients. We further demonstrate that nuclear localization of Pea3 is associated with Snail expression in breast cancer cell lines and is an independent predictor of overall survival in a Chinese breast cancer patient cohort. In conclusion, our results suggest that Pea3 may be an important prognostic marker and a therapeutic target for metastatic progression of human breast cancer.

A novel paradigm in tumor biology suggests that cancer growth is driven by stem-like cells within a tumor, called tumor-initiating cells (TICs) or cancer stem cells (CSCs). Here we describe the identification and characterization of such cells from hepatocellular carcinoma (HCC) using the marker CD133. CD133 accounts for approximately 1.3%-13.6% of the cells in the bulk tumor of human primary HCC samples. When compared with their CD133⁻ counterparts, CD133(+) cells not only possess the preferential ability to form undifferentiated tumor spheroids in vitro but also express an enhanced level of stem cell-associated genes, have a greater ability to form tumors when implanted orthotopically in immunodeficient mice, and can be serially passaged into secondary animal recipients. Xenografts resemble the original human tumor and maintain a similar percentage of tumorigenic CD133(+) cells. Quantitative PCR analysis of 41 separate HCC tissue specimens with follow-up data found that CD133(+) tumor cells were frequently detected at low quantities in HCC, and their presence was also associated with worse overall survival and higher recurrence rates. Subsequent differential microRNA expression profiling of CD133(+) and CD133⁻ cells from human HCC clinical specimens and cell lines identified an overexpression of miR-130b in CD133(+) TICs. Functional studies on miR-130b lentiviral-transduced CD133⁻ cells demonstrated superior resistance to chemotherapeutic agents, enhanced tumorigenicity in vivo, and a greater potential for self renewal. Conversely, antagonizing miR-130b in CD133(+) TICs yielded an opposing effect. The increased miR-130b paralleled the reduced TP53INP1, a known miR-130b target. Silencing TP53INP1 in CD133⁻ cells enhanced both self renewal and tumorigenicity in vivo. Collectively, miR-130b regulates CD133(+) liver TICs, in part, via silencing TP53INP1.

One of the common features in advanced prostate cancer is bone metastasis. In this study, we investigated the clinical relevance of a bone factor, MSX2, in predicting the metastatic ability of prostate adenocarcinoma. Evaluation of MSX2 expression was performed using prostate cell lines as well as patient specimens. A sharp decrease in MSX2 was found in primary prostate cancer cells, 22Rv1, when compared with the non-malignant counterparts, followed by a gradual increase in more aggressive prostate cancer cell lines. Interestingly, the MSX2 protein was upregulated and predominantly expressed in the nucleus in aggressive prostate cancer cell line, C4-2b, compared with the less aggressive 22Rv1. Consistent with the in vitro results, MSX2 nuclear expression was significantly higher in nodular hyperplasia when compared with high-grade prostatic intraepithelial neoplasia (PIN), while MSX2 nuclear expression in prostate adenocarcinoma was higher than that in high-grade PIN. Importantly, MSX2 expression was increased significantly in tumors with metastasis compared with those without metastasis. Finally, MSX2 nuclear scores were significantly increased in patients with preoperative serum PSA >20 ng/mL. No correlation between MSX2 nuclear score and Gleason score was found. Taken together, MSX2 may serve as a potential biomarker in predicting primary prostate tumors with higher metastatic capability.

ADAMTS metalloprotease family member ADAMTS9 maps to 3p14.2 and shows significant associations with the aerodigestive tract cancers esophageal squamous cell carcinoma (ESCC) and nasopharyngeal carcinoma (NPC). However, the functional impact of ADAMTS9 on cancer development has not been explored. In this study, we evaluated the hypothesized antiangiogenic and tumor-suppressive functions of ADAMTS9 in ESCC and NPC, in stringent tumorigenicity and Matrigel plug angiogenesis assays. ADAMTS9 activation suppressed tumor formation in nude mice. Conversely, knockdown of ADAMTS9 resulted in clones reverting to the tumorigenic phenotype of parental cells. In vivo angiogenesis assays revealed a reduction in microvessel numbers in gel plugs injected with tumor-suppressive cell transfectants. Similarly, conditioned medium from cell transfectants dramatically reduced the tube-forming capacity of human umbilical vein endothelial cells. These activities were associated with a reduction in expression levels of the proangiogenic factors MMP9 and VEGFA, which were consistently reduced in ADAMTS9 transfectants derived from both cancers. Taken together, our results indicate that ADAMTS9 contributes an important function in the tumor microenvironment that acts to inhibit angiogenesis and tumor growth in both ESCC and NPC. ©2010 AACR., link_to_OA_fulltext

Background: Whether the TNM staging system is applicable after neoadjuvant chemoradiation in esophageal cancer is controversial. The aim of this study was to evaluate the prognostic value of histopathological regression of the primary tumor in postchemoradiated patients. Materials and Methods: The pretherapeutic and pathological ypTNM stages of patients who have had neoadjuvant chemoradiation followed by esophagectomy were analyzed. The percentage of residual viable cells of the primary tumor (ypV) and other clinicopathological factors were tested for their prognostic value. Results: Of 175 recruited patients, 55 (31.4%) achieved pathological complete response. The median survival of these 55 patients was significantly longer than those with other disease stages (124.8 vs 21.1 months) (P, published_or_final_version, Springer Open Choice, 01 Dec 2010

Background: Id-1 is overexpressed in and correlated with metastatic potential of prostate cancer. The role of Id-1 in this metastatic process was further analysed. Methods: Conditioned media from prostate cancer cells, expressing various levels of Id-1, were used to stimulate pre-osteoclast differentiation and osteoblast mineralisation. Downstream effectors of Id-1 were identified. Expressions of Id-1 and its downstream effectors in prostate cancers were studied using immunohistochemistry in a prostate cancer patient cohort (N=110). Results: We found that conditioned media from LNCaP prostate cancer cells overexpressing Id-1 had a higher ability to drive osteoclast differentiation and a lower ability to stimulate osteoblast mineralisation than control, whereas conditioned media from PC3 prostate cancer cells with Id-1 knockdown were less able to stimulate osteoclast differentiation. Id-1 was found to negatively regulate TNF-β and this correlation was confirmed in human prostate cancer specimens (P=0.03). Furthermore, addition of recombinant TNF-β to LNCaP Id-1 cell-derived media blocked the effect of Id-1 overexpression on osteoblast mineralisation. Conclusion: In prostate cancer cells, the ability of Id-1 to modulate bone cell differentiation favouring metastatic bone disease is partially mediated by TNF-β, and Id-1 could be a potential therapeutic target for prostate cancer to bone metastasis.

Our previous studies and those of others have indicated that X-linked inhibitor of apoptosis protein (XIAP) holds promise as a target gene in colon cancer gene therapy. In this study, we constructed an adenoviral vector to deliver small hairpin RNA (shRNA) against XIAP (XIAP-shRNA) into colon cancer cells and tested its therapeutic efficacy in vitro and in vivo. We first confirmed an overexpression of XIAP in colon cancer cells and human cancer tissues. We then designed XIAP-small interfering RNA (siRNA) and confirmed the knockdown effect of these siRNAs in colon cancer cells. The sequences of the effective siRNAs were converted into shRNA and then packed into replication-deficient adenoviral vectors using BLOCK-iT Adenoviral RNAi Expression System to generate Adv-XIAP-shRNA. Infection of HT29 and HCT116 cells with Adv-XIAP-shRNA led to enhanced caspase-3 activity, which was associated with increased apoptosis and reduced cell proliferation. The therapeutic effect of Adv-XIAP-shRNA was then tested in xenograft tumors in nude mice. We showed that treatment of the xenograft tumors derived from HCT116 cells with Adv-XIAP-shRNA resulted in a retardation of tumor growth, which was associated with enhanced apoptosis, increased caspase-3 activity, and reduced expression of proliferating cell nuclear antigen in the tumor tissues. Treatment of xenograft tumors with Adv-XIAP-shRNA did not affect the expressions of inflammatory cytokines in tumor-bearing mice. Thus, Adv-XIAP-shRNA–mediated down-regulation of XIAP exerts a therapeutic effect in colon cancer by promoting apoptosis and inhibiting proliferation of colon cancer cells, and the antitumor effect of Adv-XIAP-shRNA was unlikely to be related to virus-induced immune response. [Mol Cancer Ther 2009;8(9):2762–70]

Androgen receptor (AR) is a ligand-dependent transcription factor and its activity is regulated by numerous AR coregulators. Aberrant expression of AR coregulators in prostate cancer cells has an important role in the development and progression of prostate cancer. We report here that CDC25A, a cell cycle-promoting phosphatase over-expressed in a number of cancers, functions as an AR coregulator suppressing the AR transcriptional activity. In this study, we found that CDC25A is upregulated in human prostate cancer and its expression level is positively associated with the Gleason score and disease metastasis. More importantly, we showed that CDC25A can physically interact with AR through its putative catalytic domain. In addition, ectopic expression of CDC25A in prostate cancer cell lines suppresses PSA and Probasin promoter activities significantly, indicating that CDC25A may function as an AR corepressor. This was further confirmed by knockdown of endogenous CDC25A expression using small interfering RNA (siRNA), which resulted in upregulation of PSA promoter activity. Moreover, a truncated mutant that does not interact with AR fails to suppress the PSA promoter activity, indicating that CDC25A downregulates androgen-responsive promoter by physically interacting with AR. Taken together, our results demonstrated a novel function of CDC25A in the regulation of androgen signaling in human prostate cancer cells.

Recent studies have revealed an oncogenic role of DJ-1 through its ability to transform normal cells, prevent oxidative damage, and inhibit apoptosis. However, its role in esophageal squamous cell carcinoma (ESCC) is unknown. In this study, by immunohistochemistry, we analyzed the expression of DJ-1 in 81 ESCC tumors, 31 paired nonneoplastic esophageal epithelia, and 19 paired ESCC lymph node metastases. We found that cytoplasmic DJ-1 expression was significantly higher in ESCC and ESCC lymph node metastases than in nonneoplastic esophageal epithelium. ESCC specimens with high distant metastatic potential also had a significantly higher level of nuclear DJ-1 expression (P = 0.018). By Kaplan-Meier analysis, we found that a high level of nuclear DJ-1 was significantly associated with poorer patient survival in our cohort (P = 0.028). To investigate whether DJ-1 promotes ESCC progression through phosphatidylinositol 3-kinase pathway and modulation of apoptosis, we performed immunohistochemistry of pAkt and Daxx. We found that DJ-1 expression was significantly associated with pAkt, whereas nuclear DJ-1 expression was significantly correlated with nuclear expression of Daxx. These results suggest that phosphatidylinositol 3-kinase pathway and Daxx-regulated apoptosis might be important in DJ-1-mediated ESCC progression. By using multivariate Cox regression, we further showed that T4 stage (P = 0.003) and DJ-1 (P = 0.034) are independent predictors of patient survival. In conclusion, our results suggest that DJ-1 plays a very important role in transformation and progression of ESCC and may be used as a prognostic marker in ESCC. (Cancer Epidemiol Biomarkers Prev 2008;17(12):3593–602)

Recent research efforts in stem cell and cancer biology have put forth a "stem cell model of carcinogenesis" which stipulates that the capability to maintain tumor formation and growth specifically resides in a small population of cells called cancer stem cells. The stem cell-like characteristics of these cells, including their ability to self-renew and differentiate; and their limited number within the bulk of the tumor mass, are believed to account for their capability to escape conventional therapies. In the past few years, the hypothesis of stem cell-driven tumorigenesis in liver cancer has received substantial support from the recent ability to identify and isolate a subpopulation of liver cancer cells that is not only able to initiate tumor growth, but also serially establish themselves as tumor xenografts with high efficiency and consistency. In this review, stem cell biology that contributes to explain tumor development in the particular context of liver cancer will be discussed. We will begin by briefly considering the knowledge available on normal liver stem cells and their role in tissue renewal and regeneration. We will then summarize the current scientific knowledge of liver cancer stem cells, discuss their relevance to the diagnosis and treatment of the disease and consider the outstanding challenges and potential opportunities that lie ahead of us.

SUMMARY: Aim: The ever-growing number and increasing survival of haematopoietic stem cell transplantation (HSCT) allow better recognition of its associated renal injuries. We aimed to study the clinicopathologic features of renal biopsies after HSCT by reviewing 13 percutaneous renal biopsies in our institute (Queen Mary Hospital). Methods: A retrospective clinicopathologic study of all renal biopsies archived to the Department of Pathology, Queen Mary Hospital during the period January 1999 to December 2006 was performed. Biopsies from patients with HSCT were selected. Clinical data on presentation and follow up were retrieved from hospital records and physicians. Results: In the 8-year period, a total of 2233 native renal biopsies were archived. Thirteen renal biopsies were selected from 12 patients with HSCT (11 allogeneic, one autologous). All but one patient were male. The age at renal biopsy ranged from 7 to 63 years (median: 32 years). The median interval of renal biopsy after HSCT was 24 months (range 1–134 months). Evidence of graft-versus-host disease was found in nine patients. The most common presentation was significant proteinuria (10 cases) and renal impairment (eight cases). The predominant histological changes were membranous glomerulonephritis (n = 4) and thrombotic microangiopathy (n = 4). One case of focal segmental glomerulosclerosis, IgA nephropathy, minimal change disease, acute tubular necrosis and hypertensive nephrosclerosis were also recorded. Four of our patients died at 0–11 months after renal biopsy. Of the remaining eight patients with a mean follow up of 43.6 months (range, 10–98 months), chronic renal impairment were found in three (37.5%) patients and significant proteinuria also persisted in three. One patient had cytogenetic evidence of relapse of underlying haematological malignancy after HSCT. Conclusion: Among the various renal lesions after HSCT, membranous glomerulonephritis and thrombotic microangiopathy were the most common. Mechanisms of renal injury varied from graft-versus-host disease-associated immune complex deposition to non-immune complex injury on endothelial cells, glomerular epithelial cells and tubular epithelium. Pathologists and clinicians should attend to the histological and temporal heterogeneity of renal injury when managing patients after HSCT.

TWIST, a helix-loop-helix transcription factor, is highly expressed in many types of human cancer. We have previously found that TWIST confers prostate cancer cells with an enhanced metastatic potential through promoting epithelial-mesenchymal transition (EMT) and a high TWIST expression in human prostate cancer is associated with an increased metastatic potential. The predilection of prostate cancer cells to metastasize to bone may be due to two interplaying mechanisms (i) by increasing the rate of bone remodeling and (ii) by undergoing osteomimicry. We further studied the role of TWIST in promoting prostate cancer to bone metastasis. TWIST expression in PC3, a metastatic prostate cancer cell line, was silenced by small interfering RNA and we found that conditioned medium from PC3 with lower TWIST expression had a lower activity on stimulating osteoclast differentiation and higher activity on stimulating osteoblast mineralization. In addition, we found that these effects were, at least partly, associated with TWIST-induced expression of dickkopf homolog 1 (DKK-1), a factor that promotes osteolytic metastasis. We also examined TWIST and RUNX2 expressions during osteogenic induction of an organ-confined prostate cancer cell, 22Rv1. We observed increased TWIST and RUNX2 expressions upon osteogenic induction and downregulation of TWIST through short hairpin RNA reduced the induction level of RUNX2. In summary, our results suggest that, in addition to EMT, TWIST may also promote prostate cancer to bone metastasis by modulating prostate cancer cell-mediated bone remodeling via regulating the expression of a secretory factor, DKK-1, and enhancing osteomimicry of prostate cancer cells, probably, via RUNX2.

Missing in metastasis (MIM) proteins are important regulators in controlling cell growth and development. There has been accumulating evidence suggesting a role of MIM-B in carcinogenesis, yet its role in the development of hepatocellular carcinoma has not been examined thus far. In this study, we investigated the clinicopathological significance of MIM-B in tumor and its matched adjacent nontumor tissue obtained from 40 patients with hepatocellular carcinoma. Increased MIM-B messenger RNA and protein expression, as detected by quantitative real-time polymerase chain reaction and Western blot, respectively, was found in hepatocellular carcinoma clinical samples; and its expression was significantly associated with early pathologic tumor-node-metastasis stage group (P = .007), presence of tumor encapsulation (P = .034), and absence of venous infiltration (P = .038). Higher levels of MIM-B expression were found to be associated with early stage disease. Elevated MIM-B expression may influence the development of hepatocellular carcinoma and may possibly be a powerful indicator for the disease at an early stage.

Background & Aims: Recent efforts in stem cell biology suggest that tumors are organized in a hierarchy of heterogeneous cell populations and that the capability to maintain tumor formation/growth specifically resides in a small population of cells called cancer stem cells (CSCs). The aim of this study is to identify, isolate, and characterize the CSC population that drives and maintains hepatocellular carcinoma (HCC) growth and metastasis. Methods: Normal stem cells involved in liver regeneration were identified using a severe partial hepatectomy model. Purified HCC cells, with or without expression of the identified normal stem cell phenotype, were evaluated, based on their tumorigenic potential and exhibition of defined stem/progenitor cell-like properties, to determine whether liver CSCs can be or partly be identified by this surface marker. Results: We report the identification and isolation of a population of CSCs expressing a CD133 surface phenotype from human liver cell lines. CD133 + cells possess a greater colony-forming efficiency, higher proliferative output, and greater ability to form tumor in vivo. These cells are endowed with characteristics similar to those of progenitor cells including the expression of "stemness" genes, the ability to self-renew, and the ability to differentiate into nonhepatocyte-like lineages. Furthermore, CD133 is found to represent only a minority of the tumor cell population in human HCC specimens. Conclusions: We report the identification of a CSC population in HCC characterized by their CD133 phenotype. The identification of tumorigenic liver CSCs could provide new insight into the HCC tumorigenic process and possibly bear great therapeutic implications.