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Supplementary Table S1 from Aldehyde Dehydrogenase Discriminates the CD133 Liver Cancer Stem Cell Populations

Recent efforts in our study of cancer stem cells (CSC) in hepatocellular carcinoma (HCC) have led to the identification of CD133 as a prominent HCC CSC marker. Findings were based on experiments done on cell lines and xenograft tumors where expression of CD133 was detected at levels as high as 65%. Based on the CSC theory, CSCs are believed to represent only a minority number of the tumor mass. This is indicative that our previously characterized CD133+ HCC CSC population is still heterogeneous, consisting of perhaps subsets of cells with differing tumorigenic potential. We hypothesized that it is possible to further enrich the CSC population by means of additional differentially expressed markers. Using a two-dimensional PAGE approach, we compared protein profiles between CD133+ and CD133− subpopulations isolated from Huh7 and PLC8024 and identified aldehyde dehydrogenase 1A1 as one of the proteins that are preferentially expressed in the CD133+ subfraction. Analysis of the expression of several different ALDH isoforms and ALDH enzymatic activity in liver cell lines found ALDH to be positively correlated with CD133 expression. Dual-color flow cytometry analysis found the majority of ALDH+ to be CD133+, yet not all CD133+ HCC cells were ALDH+. Subsequent studies on purified subpopulations found CD133+ALDH+ cells to be significantly more tumorigenic than their CD133−ALDH+ or CD133−ALDH− counterparts, both in vitro and in vivo. These data, combined with those from our previous work, reveal the existence of a hierarchical organization in HCC bearing tumorigenic potential in the order of CD133+ALDH+ > CD133+ALDH− > CD133−ALDH−. ALDH, expressed along CD133, can more specifically characterize the tumorigenic liver CSC population. (Mol Cancer Res 2008;6(7):1146–53)

Representative immunohistochemical staining of high, medium and low FSTL1 in non-neoplastic squamous epithelium (non-tumor) and poorly differentiated ESCC tissue from 6 different patients.

FSTL1(+) cells in ESCC clinical samples that received (with treatment, n = 22) or did not receive (without treatment, n = 15) chemotherapy treatment prior to resection, as detected by IHC analyses.

Quantification of number of cells that migrated through a membrane, invaded through a Matrigel-coated membrane, formed spheroids or formed colonies in (A) KYSE150-Luc cells co-cultured with conditioned medium collected from EV or FSTL1 OE cells; or (B) KYSE150-Luc cells treated with control or recombinant FSTL1, in the presence or absence of IMD-0354.

Quantification of number of cells that migrated through a membrane, invaded through a Matrigel-coated membrane, formed spheroids or formed colonies in (A) KYSE150-Luc cells co-cultured with conditioned medium collected from EV or FSTL1 OE cells; or (B) KYSE150-Luc cells treated with control or recombinant FSTL1, in the presence or absence of C34.

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.

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Representative images and quantification of number of cells that migrated or invaded in EC109 cells with or without FSTL1 suppressed and KYSE150-Luc cells with or without FSTL1 overexpressed, in the presence of mitomycin C.

Purpose: Previously, FTY720 was found to possess potent anticancer effects on various types of cancer. In the present study, we aimed to first verify the role of Runx2 in prostate cancer progression and metastasis, and, subsequently, assessed if FTY720 could modulate Runx2 expression, thus interfering downstream events regulated by this protein.Experimental Design: First, the association between Runx2 and prostate cancer progression was assessed using localized prostate cancer specimens and mechanistic investigation of Runx2-induced cancer aggressiveness was then carried out. Subsequently, the effect of FTY720 on Runx2 expression and transcriptional activity was investigated using PC-3 cells, which highly expressed Runx2 protein. Last, the involvement of Runx2 in FTY720-induced anticancer effects was evaluated by modulating Runx2 expression in various prostate cancer cell lines.Results: Runx2 nuclear expression was found to be up-regulated in prostate cancer and its expression could be used as a predictor of metastasis in prostate cancer. Further mechanistic studies indicated that Runx2 accelerated prostate cancer aggressiveness through promotion of cadherin switching, invasion toward collagen I, and Akt activation. Subsequently, we found that FTY720 treatment down-regulated Runx2 expression and its transcriptional activity, as well as inhibited its regulated downstream events. More importantly, silencing Runx2 in PC-3 enhanced FTY720-induced anticancer effects as well as cell viability inhibition, whereas overexpressing Runx2 in 22Rv1 that expressed very low endogenous Runx2 protein conferred resistance in the same events.Conclusion: This study provided a novel mechanism for the anticancer effect of FTY720 on advanced prostate cancer, thus highlighting the therapeutic potential of this drug in treating this disease.

Esophageal squamous cell carcinoma (ESCC) has a generally poor prognosis, and molecular markers to improve early detection and predict outcomes are greatly needed. Here, we report that the BMP-binding follistatin-like protein FSTL1 is overexpressed in ESCCs, where it correlates with poor overall survival. Genetic amplification of FSTL1 or chromosome 3q, where it is located, occurred frequently in ESCC, where FSTL1 copy number correlated positively with higher FSTL1 protein expression. Elevating FSTL1 levels by various means was sufficient to drive ESCC cell proliferation, clonogenicity, migration, invasion, self-renewal, and cisplatin resistance in vitro and tumorigenicity and distant metastasis in vivo. Conversely, FSTL1 attenuation by shRNA or neutralizing antibody elicited the opposite effects in ESCC cells. mRNA profiling analyses suggested that FSTL1 drives ESCC oncogenesis and metastasis through various pathways, with deregulation of NFκB and BMP signaling figuring prominently. Cross-talk between the NFκB and BMP pathways was evidenced by functional rescue experiments using inhibitors of NFκB and TLR4. Our results establish the significance of FSTL1 in driving oncogenesis and metastasis in ESCC by coordinating NFκB and BMP pathway control, with implications for its potential use as a diagnostic or prognostic biomarker and as a candidate therapeutic target in this disease setting. Cancer Res; 77(21); 5886–99. ©2017 AACR.

Supplementary Data from Suppression of Androgen-Independent Prostate Cancer Cell Aggressiveness by FTY720: Validating Runx2 as a Potential Antimetastatic Drug Screening Platform

Suppl Figure Legends 1-5 and Tables showing clinico-pathological correlation of endogenous and secretory FSTL1 as well as pathway analysis of ESCC cells with or without FSTL1 overexpressed.

PDF file - 1419KB Supplementary Fig S1. Human growth factor antibody array analysis of the conditioned media from HKESC-3-CON and HKESC-3-Id1 cells. Supplementary Fig S2. Effect of knockdown Id1 expression on IGF2 level using a second RNAi sequence. Supplementary Fig S3. Effect of ectopic Id1 expression on IGF2 level in multiple cancer types. Supplementary Fig S4. Effect of Id1 manipulation on IGF2 mRNA expression in ESCC cells. Supplementary Fig S5. IGF2, Id1, and p-AKT expressions in human esophageal cancer. Supplementary Fig S6. Western blot analysis of Id1 and IGF2 expressions in ESCC cell lines. Supplementary Fig S7. Effects of Id1 knockdown on autocrine stimulation of esophageal cancer cell proliferation, migration, and survival. Supplementary Fig S8. Ki-67 and TUNEL staining of tumor xenografts from three groups of mice inoculated with KYSE150-CON-shCON, KYSE150-Id1-shCON, and KYSE150-Id1- shIGF2 cells, respectively. Supplementary Fig S9. Ki-67 and TUNEL staining in "responder" tumors of different groups of mice bearing KYSE150-CON-shCON, KYSE150-Id1-shCON, and KYSE150-Id1- shIGF2 "instigating" tumors, respectively. Supplementary Fig S10. Anti-tumor effects of IGF2-neutralizing antibody in vivo. Supplementary Fig S11. Immunohistochemical analysis of tumor xenografts treated with cixutumumab or the isotype IgG. Supplementary Fig S12. TUNEL staining of tumor xenografts from two groups of mice inoculated with KYSE150 (A) and KYSE270 (B) respectively. Supplementary Fig S13. Evaluation of toxic effects of cixutumumab in nude mice. A, comparison of body weight of cixutumumab-treated mice and isotype IgG-treated control mice. B, histological examination of lung, liver and kidney specimens (H&E stained). Supplementary Fig S14. Increased thymidylate synthase expression in FR cells was inhibited by cixutumumab.

PDF file - 8KB, Immunohistochemical analysis of expression levels of Id1, IGF2, and p-AKT in human ESCC specimens.

PDF file - 490K, Supplemental Figure 1. A significantly deregulated network of genes in CD90+ esophageal T-ICs identified by genome-wide expression profiling and Ingenuity Pathway Analysis (IPA).

Supplementary Table 2 from MicroRNA-616 Induces Androgen-Independent Growth of Prostate Cancer Cells by Suppressing Expression of Tissue Factor Pathway Inhibitor TFPI-2

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. Cancer Res; 70(13); 5567–76. ©2010 AACR.

Expression of microRNA genes is profoundly altered in cancer but their role in the development of androgen-independent prostate cancer has received limited attention as yet. In this study, we report a functional impact in prostate cancer cells for overexpression of the microRNA miR-616, which occurred consistently in cells that were androgen-independent (AI) versus androgen-dependent (AD). miR-616 overexpression was confirmed in malignant prostate tissues as opposed to benign prostate specimens. Stable miR-616 overexpression in LNCaP cells by a lentiviral-based approach stimulated AI prostate cancer cell proliferation in vitro whereas concomitantly reducing androgen-induced cell growth. More importantly, miR-616 overexpressing LNCaP cells overcame castration resistance as shown by an enhanced ability to proliferate in vivo after bilateral orchiectomy. Conversely, antagonizing miR-616 in AI prostate cancer cells yielded opposite effects. Microarray profiling and bioinformatics analysis identified the tissue factor pathway inhibitor TFPI-2 mRNA as a candidate downstream target of miR-616. In support of this candidacy, we documented interactions between miR-616 and the 3′UTR of TFPI-2 and determined TFPI-2 expression to be inversely correlated to miR-616 in a series of prostate cell lines and clinical specimens. Notably, reexpression of TFPI-2 in LNCaP cells with stable miR-616 overexpression rescued the AD phenotype, as shown by a restoration of androgen dependence and cell growth inhibition. Taken together, our findings define a functional involvement for miR-616 and TFPI-2 in the development and maintenance of androgen-independent prostate cancer. Cancer Res; 71(2); 583–92. ©2011 AACR.

Supplementary Table 1 from Extracellular Protease ADAMTS9 Suppresses Esophageal and Nasopharyngeal Carcinoma Tumor Formation by Inhibiting Angiogenesis

PDF file, 720K, Detection of DNA copy number change of Rab25 by dual-color fluorescent in situ hybridization (FISH) in ESCC cell lines KYSE520, EC109 and formalin-fixed paraffin-embedded ESCC clinical tissue specimens (#1 and #2) with no or low Rab25 expression. BAC probe to Rab25 and the control reference probe to the centromere of chromosome 1 are represented by red and green signals, respectively. Magnification at 1000x.

PDF file, 153K, Supplementary Materials and Methods, Tables, Figure Legends, References

DOC file - 322K, Supplemental Table 1. Primer sequences for real-time qPCR analyses. Supplemental Table 2. RPKM values of other reported CSC markers in the original 3 pairs of non-tumor (N) and ESCC (T) clinical samples (Patients #16, #18 and #19) used for RNA-Seq profiling. Supplemental Table 3. Cancer stem cell marker expression (CD90, CD44 and p75NTR / CD271) in a panel of ESCC cell lines. Supplemental Table 4. Clinicopathological correlation of CD44 and p75NTR / CD271 expression in ESCC. Supplemental Table 5. List of deregulated genes in CD90+ vs. CD90- cells in ESCC cells KYSE140 (cut off ≥ 3 folds).

PDF file - 1414K, Supplemental Figure 2. CD90, CD44 and p75NTR / CD271 expression in matched non-tumor (N) and primary ESCC (T) (n = 33) as detected by real-time qPCR.

Supplementary Figure Legend from MicroRNA-616 Induces Androgen-Independent Growth of Prostate Cancer Cells by Suppressing Expression of Tissue Factor Pathway Inhibitor TFPI-2

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.

PDF file, 2272K, Expression tiling of Rab25 in 6 unpaired ESCC and non-tumor clinical samples.

Supplementary Figure 1 from MicroRNA-616 Induces Androgen-Independent Growth of Prostate Cancer Cells by Suppressing Expression of Tissue Factor Pathway Inhibitor TFPI-2

Supplementary References from MicroRNA-616 Induces Androgen-Independent Growth of Prostate Cancer Cells by Suppressing Expression of Tissue Factor Pathway Inhibitor TFPI-2

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.

The current study reveals the clinicopathological association of PD-L1 in Hong Kong esophageal squamous cell carcinoma (ESCC) patients and the differential regulation of PD-L1 by standard first-line chemotherapy in ESCC. Immunohistochemical analysis of tissue microarray data from 84 Hong Kong ESCC patients shows that PD-L1 was expressed in 21% of the tumors. Positive PD-L1 staining was significantly associated with later disease stage (stages III and IV) (P value = .0379) and lymph node metastasis (P value = .0466) in the Hong Kong cohort. Furthermore, PD-L1 expression was significantly induced in ESCC cell lines after standard chemotherapy treatments, along with EGFR and ERK activation in both in vitro studies and the in vivo esophageal orthotopic model. The endogenous expression of PD-L1 was reduced by treatment with an EGFR inhibitor (erlotinib) or by the knockdown of EGFR. Moreover, the upregulation of PD-L1 by chemotherapy was also attenuated by the treatment with erlotinib and a MAPK/MEK inhibitor (AZD6244), suggesting that PD-L1 is regulated by the EGFR/ERK pathway in ESCC. The regulation of PD-L1 by the EGFR pathway was further supported by the correlation of PD-L1 and EGFR expression observed in the commercially available tissue microarray set (P value = .028). Taken together, the current study was the first to demonstrate the upregulation of PD-L1 by chemotherapy in ESCC and its regulation through the EGFR/ERK pathway. The results suggest the potential usefulness of combined conventional chemotherapy together with anti-PD-L1 immunotherapy to achieve better treatment outcome.

Introduction Over the past 17 years (2002–2018), 898,831 people participated in the Hong Kong Standard Chartered Marathon (HKSCM). Purpose We aim to study the incidence of marathon-related sudden cardiac arrest (SCA) and non-fatal myocardial injury (NFMI). Methods All the HKSCM related admissions between 2002 and 2017 were retrieved from the accident and emergency department admission records. We reviewed the data of HKSCM runners admitted for SCA and NFMI. Results Fourteen and thirty-nine runners were admitted for SCA (Mean age 38±13 years-old, 12 men) and NFMI (Mean age 42±12 years-old; 34 men) respectively. Among the SCA runners, the arrest rhythms could not be retrieved in 3 cases. In the remaining 11 SCA runners, the arrest rhythms were ventricular fibrillation (VF) (N=6), pulseless-electrical activity (PEA) (N=3), and asystole (N=2). Nine of the 14 SCA runners were successfully resuscitated. Only 2 out of the 14 SCA runners had significant atherosclerotic coronary artery disease (CAD) requiring coronary angioplasty. Among the SCA runners, the etiologies of cardiac arrest were myocardial infarction (MI) (N=2), ischemic cardiomyopathy with VF (N=1), idiopathic VF (N=3), malignant coronary anomaly (N=1), and idiopathic (N=5). Percutaneous coronary intervention (PCI) was performed in 2 SCA runner with MI. Implantable cardioverter-defibrillators were implanted in 1 resuscitated runner with VF. Postmortem examination of the 3 deceased runners showed significant CAD in two and was unrevealing in one. Among the 39 NFMI runners, coronary angiograms (CAG) or CT coronary angiogram were performed in 14 cases (36%), which showed minor CAD or unremarkable findings in 7 runners, and significant CAD in 7 runners. PCI and coronary artery bypass were performed in 5 and 2 NFMI runners respectively. Only 8 out of 39 NFMI runners reported chest pain. Significant ischemic ECG changes were detected in 9 out of 39 NFMI runners. Invasive CAGs were not performed in the remaining NFMI runners due to low pre-test likelihood of CAD and normal non-invasive test results. Conclusions The incidence of SCA and mortality among HKSCM runners was 1.56 per 100,000 and 0.56 per 100,000 respectively. The incidence of NFMI was 4.3 per 100,000. Coronary artery disease, coronary anomaly and idiopathic VF were the commonest etiologies of SCA.

Multidrug resistance (MDR) is one of conventional cancer chemotherapy&rsquo, s limitations. Our group previously synthesized a series of quinoline-based compounds in an attempt to identify novel anticancer agents. With a molecular docking analysis, the novel compound 160a was predicted to target p-glycoprotein, an MDR candidate. The purpose of this study is to evaluate 160a&rsquo, s MDR reversal effect and investigate the underlying mechanism at the molecular level. To investigate 160a&rsquo, s inhibitory effect, we used a series of parental cancer cell lines (A549, LCC6, KYSE150, and MCF-7), the corresponding doxorubicin-resistant cell lines, an MTS cytotoxicity assay, an intracellular doxorubicin accumulation test, and multidrug resistance assays. The Compusyn program confirmed, with a combination index (CI) value greater than 1, that 160a combined with doxorubicin exerts a synergistic effect. Intracellular doxorubicin accumulation and transported calcein acetoxymethyl (AM) (a substrate for p-glycoprotein) were both increased when cancer cells with MDR were treated with compound 160a. We also showed that compound 160a&rsquo, s MDR reversal effect can persist for at least 1 h. Taken together, these results suggest that the quinoline compound 160a possesses high potential to reverse MDR by inhibiting p-glycoprotein-mediated drug efflux in cancer cells with MDR.

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.

Purpose This study aims to examine the expression profiles miR-1288 in oesophageal squamous cell carcinoma (ESCC). The cellular implications and target interactions of ESCC cells following miR-1288 overexpression was also examined. Methods In total, 120 oesophageal tissues (90 primary ESCCs and 30 non-neoplastic tissues) were recruited for miR-1288 expression analysis using qRT-PCR. An exogenous miR-1288 mimic and its inhibitor were used to explore the in-vitro effects of miR-1288 on ESCC cells by performing cell proliferation, colony formation, cell invasion and migration assays. Localisation and modulatory changes of various miR-1288 regulated proteins such as FOXO1, p53, TAB3, BCL2 and kRAS was examined using immunofluorescence and western blot. Results Overexpression of miR-1288 was more often noted in ESCC tissues when compared to non-neoplastic oesophageal tissues. High expression was often noted in high grade carcinomas and with metastases. Patients with high levels of miR-1288 expression showed a slightly better survival compared to patients with low miR-1288 levels. Furthermore, overexpression of miR-1288 showed increased cell proliferation and colony formation, improved cell migration and enhanced cell invasion properties in ESCC cells. In addition, miR-1288 overexpression in ESCC cells showed repression of cytoplasmic tumour suppressor FOXO1 protein expression. Inversely, inhibition of miR-1288 expression exhibited remarkable upregulation of FOXO1 protein, while expressions of other tested proteins remain unchanged. Conclusions Up regulation of miR-1288 expression in ESCC tissues and miR-1288 induced oncogenic features of ESCC cells in-vitro indicates the oncogenic roles of miR-1288 in ESCCs. Overexpression of miR-1288 play a key role in the pathogenesis of ESCCs and its modulation may have potential therapeutic value in patients with ESCC.

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.