A novel and sensitive DNA methylation marker for the urine-based liquid biopsies to detect bladder cancer.

BACKGROUND: Better prognostic outcome is closely correlated with early detection of bladder cancer. Current non-invasive urianalysis relies on simultaneously testing multiple methylation markers to achieve relatively high accuracy. Therefore, we have developed an easy-to-use, convenient, and accurate single-target urine-based DNA methylation test for the malignancy. METHODS: By analyzing TCGA data, 344 candidate markers with 424 primer pairs and probe sets synthesized were systematically screened in cancer cell lines, paired tissue specimens, and urine sediments from bladder cancer patients and normal controls. The identified marker was further validated in large case-control cohorts. Wilcoxon rank sum tests and c2 tests were performed to compare methylation levels between case-control groups and correlate methylation levels with demographic and clinical characteristics. In addition, MSP, qMSP, RT-PCR, western blot analysis, and immunohistochemistry were performed to measure levels of DNA methylation, mRNA transcription, and protein expression in cancer cell lines and tissues. RESULTS: A top-performing DMRTA2 marker identified was tested in both discovery and validation sets, showing similar sensitivity and specificity for bladder cancer detection. Overall sensitivity in the aggregate set was 82.9%(179/216). The specificity, from a control group consisting of patients with lithangiuria, prostatoplasia, and prostatitis, is 92.5%(468/506). Notably, the methylation assay had the highest sensitivities for tumors at stages of T1(90.4%) and T2(95.0%) compared with Ta (63.0%), T3(81.8%), and T4(81.8%). Furthermore, the test showed admirable detection rate of 80.0%(24/30) for recurring cancers. While methylation was observed in 39/54(72.2%) urine samples from patients with carcinomas of renal pelvis and ureter, it was detected at extremely low rate of 6.0%(8/133) in kidney and prostate cancers. Compared with SV-HUC-1, the normal bladder epithelial cell line, DMRTA2 was hypermethylated in 8/9 bladder cancer cell lines, consistent with the results of MSP and qMSP, but not correlated with mRNA and protein expression levels in these cell lines. Similarly, DMRTA2 immunostaining was moderate in some tissues but weak in others. Further studies are needed to address functional implications of DMRTA2 hypermethylation. CONCLUSIONS: Our data demonstrated that a single-target DNA methylation signature, mDMRTA2, could be highly effective to detect both primary and recurring bladder cancer via urine samples.

High-Yield Methylation Markers for Stool-Based Detection of Colorectal Cancer.

BACKGROUND: Many methylation markers associated with colorectal cancer have been reported, but few of them are actually used in clinical practice. AIMS: This study was designed to identify promising methylation markers for stool-based detection of colorectal cancer. METHODS: We first tested 324 reported methylated genes in colorectal cancer cell lines. A total of 111 heavily methylated genes were selected for further evaluation with a pilot set of colorectal cancer and adjacent normal tissues. Ten high-yield methylated markers were further studied in 319 tissue samples. Eventually, the four best markers, namely methylated COL4A1, COL4A2, TLX2, and ITGA4, were validated in 240 stool samples. Methylation-specific PCR (MSP) and real-time MSP (qMSP) were employed for methylation detection. RESULTS: After hierarchical selection, ten differentially methylated genes demonstrated high sensitivity and specificity for the detection of colorectal cancer in tissue. When validated in stool samples, the four with the best performance-COL4A1, COL4A2, TLX2, and ITGA4-were able to detect 82.5-92.5% of colorectal cancers and 41.6-58.4% of adenomas (≥ 1 cm) with specificity of 88.0-96.4%. The best combination, COL4A2 and TLX2, detected 91.3% of CRCs and 51.9% of advanced adenomas in stool with 97.6% specificity. CONCLUSIONS: Methylated COL4A1, COL4A2, TLX2, and ITGA4 demonstrated high accuracy for the detection of colorectal neoplasms in stool. They are potentially valuable markers for the detection of colorectal cancer.

Demographic trends and KRAS/BRAFV600E mutations in colorectal cancer patients of South China: A single-site report.

The incidence of colorectal cancer (CRC) is increasing in China. Here, we aimed to evaluate the latest demographic trends and KRAS/BRAF mutations status of Chinese CRC. Five thousand five hundred and forty-six CRC patients diagnosed from 2010 to 2017 were involved. KRAS exon 2 and BRAFV600E mutations were detected by Sanger sequencing and high-resolution melting analysis or allelic-specific probe method. Gene mutation profiles and clinicopathologic characteristics of 5495 patients were analyzed. The joinpoint regression model was used to predict the demographic data in 2018. We found KRAS exon 2 and BRAFV600E mutation rates were 37.7 and 2.8% in CRC patients. Tumors with KRAS exon 2 mutations were more likely to be present in female and patients aged older than 75 years, right colon and have well-differentiated histology. Tumors with BRAFV600E mutations were more likely to be present in the female, right colon and have poorly differentiated histology. From 2010 to 2017, the percentage of colon cancer and tubular adenocarcinoma in CRC increased substantially (from 39.3 to 51.8%, from 78.6 to 93.4%, respectively). The percentage of right colon cancer increased from 18.3 to 20.5%, which predictively may keep at 22.6% in 2018. The rise trends for patients with moderate differentiation tumor or KRAS exon 2 mutated tumor were apparent (from 50.3 to 78.6%, from 32.8 to 39.7%, respectively). In conclusion, in recent 8 years, there is a shift to the colon, especially right colon in the incidence of Chinese CRC. Moreover tubular adenocarcinoma is becoming the primary histology type.

P300 Acetyltransferase Mediates Stiffness-Induced Activation of Hepatic Stellate Cells Into Tumor-Promoting Myofibroblasts.

BACKGROUND & AIMS: Hepatic stellate cells (HSCs) contribute to desmoplasia and stiffness of liver metastases by differentiating into matrix-producing myofibroblasts. We investigated whether stiffness due to the presence of tumors increases activation of HSCs into myofibroblasts and their tumor-promoting effects, as well as the role of E1A binding protein p300, a histone acetyltransferase that regulates transcription, in these processes. METHODS: HSCs were isolated from liver tissues of patients, mice in which the p300 gene was flanked by 2 loxP sites (p300F/F mice), and p300+/+ mice (controls). The HSCs were placed on polyacrylamide gels with precisely defined stiffness, and their activation (differentiation into myofibroblasts) was assessed by immunofluorescence and immunoblot analyses for alpha-smooth muscle actin. In HSCs from mice, the p300 gene was disrupted by cre recombinase. In human HSCs, levels of p300 were knocked down with small hairpin RNAs or a mutant form of p300 that is not phosphorylated by AKT (p300S1834A) was overexpressed. Human HSCs were also cultured with inhibitors of p300 (C646), PI3K signaling to AKT (LY294002), or RHOA (C3 transferase) and effects on stiffness-induced activation were measured. RNA sequencing and chromatin immunoprecipitation-quantitative polymerase chain reaction were used to identify HSC genes that changed expression levels in response to stiffness. We measured effects of HSC-conditioned media on proliferation of HT29 colon cancer cells and growth of tumors following subcutaneous injection of these cells into mice. MC38 colon cancer cells were injected into portal veins of p300F/Fcre and control mice, and liver metastases were measured. p300F/Fcre and control mice were given intraperitoneal injections of CCl4 to induce liver fibrosis. Liver tissues were collected and analyzed by immunofluorescence, immunoblot, and histology. RESULTS: Substrate stiffness was sufficient to activate HSCs, leading to nuclear accumulation of p300. Disrupting p300 level or activity blocked stiffness-induced activation of HSCs. In HSCs, substrate stiffness activated AKT signaling via RHOA to induce phosphorylation of p300 at serine 1834; this caused p300 to translocate to the nucleus, where it up-regulated transcription of genes that increase activation of HSCs and metastasis, including CXCL12. MC38 cells, injected into portal veins, formed fewer metastases in livers of p300F/Fcre mice than control mice. Expression of p300 was increased in livers of mice following injection of CCl4; HSC activation and collagen deposition were reduced in livers of p300F/Fcre mice compared with control mice. CONCLUSIONS: In studies of mice, we found liver stiffness to activate HSC differentiation into myofibroblasts, which required nuclear accumulation of p300. p300 increases HSC expression of genes that promote metastasis.

Next-generation stool DNA test accurately detects colorectal cancer and large adenomas.

BACKGROUND & AIMS: Technical advances have led to stool DNA (sDNA) tests that might accurately detect neoplasms on both sides of the colorectum. We assessed colorectal neoplasm detection by a next-generation sDNA test and effects of covariates on test performance. METHODS: We performed a blinded, multicenter, case-control study using archived stool samples collected in preservative buffer from 252 patients with colorectal cancer (CRC), 133 with adenomas ≥ 1 cm, and 293 individuals with normal colonoscopy results (controls); two-thirds were randomly assigned to a training set and one-third to a test set. The sDNA test detects 4 methylated genes, a mutant form of KRAS, and the α-actin gene (as a reference value) using quantitative, allele-specific, real-time target and signal amplification; it also quantifies hemoglobin. We used a logistical model to analyze data. RESULTS: The sDNA test identified 85% of patients with CRC and 54% of patients with adenomas ≥1 cm with 90% specificity. The test had a high rate of detection for all nonmetastatic stages of CRC (aggregate 87% detection rate for CRC stages I-III). Detection rates increased with adenoma size: 54% ≥ 1 cm, 63% >1 cm, 77% >2 cm, 86% >3 cm, and 92% >4 cm (P < .0001). Based on receiver operating characteristic analysis, the rate of CRC detection was slightly greater for the training than the test set (P = .04), whereas the rate of adenoma detection was comparable between sets. Sensitivities for detection of CRC and adenoma did not differ with lesion site. CONCLUSIONS: Early-stage CRC and large adenomas can be detected throughout the colorectum and with high levels of accuracy by the sDNA test. Neoplasm size, but not anatomical site, affected detection rates. Further studies are needed to validate the findings in a larger population and optimize the sDNA test.

Taxonomic and functional profiling of fecal metagenomes for the early detection of colorectal cancer.

Objectives: This study aimed to identify colorectal cancer (CRC)-associated phylogenetic and functional bacterial features by a large-scale metagenomic sequencing and develop a binomial classifier to accurately distinguish between CRC patients and healthy individuals. Methods: We conducted shotgun metagenomic analyses of fecal samples from a ZhongShanMed discovery cohort of 121 CRC and 52 controls and SouthernMed validation cohort of 67 CRC and 44 controls. Taxonomic profiling and quantification were performed by direct sequence alignment against genome taxonomy database (GTDB). High-quality reads were also aligned to IGC datasets to obtain functional profiles defined by Kyoto Encyclopedia of Genes and Genomes (KEGG). A least absolute shrinkage and selection operator (LASSO) classifier was constructed to quantify risk scores of probability of disease and to discriminate CRC from normal for discovery, validation, Fudan, GloriousMed, and HongKong cohorts. Results: A diverse spectrum of bacterial and fungi species were found to be either enriched (368) or reduced (113) in CRC patients (q<0.05). Similarly, metabolic functions associated with biosynthesis and metabolism of amino acids and fatty acids were significantly altered (q<0.05). The LASSO regression analysis of significant changes in the abundance of microbial species in CRC achieved areas under the receiver operating characteristic curve (AUROCs) of 0.94 and 0.91 in the ZhongShanMed and SouthernMed cohorts, respectively. A further analysis of Fudan, GloriousMed, and HK cohorts using the same classification model also demonstrated AUROC of 0.80, 0.78, and 0.91, respectively. Moreover, major CRC-associated bacterial biomarkers identified in this study were found to be coherently enriched or depleted across 10 metagenomic sequencing studies of gut microbiota. Conclusion: A coherent signature of CRC-associated bacterial biomarkers modeled on LASSO binomial classifier maybe used accurately for early detection of CRC.

The stool DNA test is more accurate than the plasma septin 9 test in detecting colorectal neoplasia.

BACKGROUND & AIMS: Several noninvasive tests have been developed for colorectal cancer (CRC) screening. We compared the sensitivities of a multimarker test for stool DNA (sDNA) and a plasma test for methylated septin 9 (SEPT9) in identifying patients with large adenomas or CRC. METHODS: We analyzed paired stool and plasma samples from 30 patients with CRC and 22 with large adenomas from Mayo Clinic archives. Stool (n = 46) and plasma (n = 49) samples from age- and sex-matched patients with normal colonoscopy results were used as controls. The sDNA test is an assay for methylated BMP3, NDRG4, vimentin, and TFPI2; mutant KRAS; the ß-actin gene, and quantity of hemoglobin (by the porphyrin method). It was performed blindly at Exact Sciences (Madison, Wisconsin); the test for SEPT9 was performed at ARUP Laboratories (Salt Lake City, Utah). Results were considered positive based on the manufacturer's specificity cutoff values of 90% and 89%, respectively. RESULTS: The sDNA test detected adenomas (median, 2 cm; range, 1-5 cm) with 82% sensitivity (95% confidence interval [CI], 60%-95%); SEPT9 had 14% sensitivity (95% CI, 3%-35%; P = .0001). The sDNA test identified patients with CRC with 87% sensitivity (95% CI, 69%-96%); SEPT9 had 60% sensitivity (95% CI, 41%-77%; P = .046). The sDNA test identified patients with stage I-III CRC with 91% sensitivity (95% CI, 71%-99%); SEPT9 had 50% sensitivity (95% CI, 28%-72%; P = .013); for stage IV CRC, sensitivity values were 75% (95% CI, 35%-97%) and 88% (95% CI, 47%-100%), respectively (P = .56). False positive rates were 7% for the sDNA test and 27% for SEPT9. CONCLUSIONS: Based on analyses of paired samples, the sDNA test detects nonmetastatic CRC and large adenomas with significantly greater levels of sensitivity than the SEPT9 test. These findings might be used to modify approaches for CRC prevention and early detection.

Quantification of methylated markers with a multiplex methylation-specific technology.

BACKGROUND: Aberrantly methylated genes represent important markers for cancer diagnosis. We describe a multiplex detection approach to efficiently quantify these markers for clinical applications such as colorectal cancer screening. METHODS: Quantitative allele-specific real-time target and signal amplification (QuARTS) combines a polymerase-based target amplification with an invasive cleavage-based signal amplification. The fluorescence signal is detected in a fashion similar to real-time PCR. We measured the dynamic range and analytical sensitivity of multiplex QuARTS reactions with titrated plasmid DNA. We used the QuARTS technology to quantify methylated BMP3, NDRG4, VIM, and TFPI2 genes on 91 DNA samples extracted from colorectal tissues, including 37 cancers, 25 adenomas, and 29 healthy epithelia. The assays were designed in triplex format that incorporated ACTB as a reference gene. Percent methylation was calculated by dividing methylated strands over ACTB strands and multiplying by 100. RESULTS: The QuARTS method linearly detected methylated or unmethylated VIM gene down to 10 copies. No cross-reactivity was observed when methylated assays were used to amplify 10(5) copies of unmethylated gene and vice versa. The multiplex assay detected methylated genes spiked in unmethylated genes at a 0.01% ratio and vice versa. At a diagnostic specificity cutoff of 95%, methylated BMP3, NDRG4, VIM, and TFPI2 detected 84%, 92%, 86%, and 92% of colorectal cancers and 68%, 76%, 76%, and 88% of adenomas, respectively. CONCLUSIONS: The QuARTS technology provides a promising approach for quantifying methylated markers. The markers assayed highly discriminated colorectal neoplasia from healthy epithelia.

Downregulated cytotoxic CD8+ T-cell identifies with the NKG2A-soluble HLA-E axis as a predictive biomarker and potential therapeutic target in keloids.

Keloids are an abnormal fibroproliferative wound-healing disease with a poorly understood pathogenesis, making it difficult to predict and prevent this disease in clinical settings. Identifying disease-specific signatures at the molecular and cellular levels in both the blood circulation and primary lesions is urgently needed to develop novel biomarkers for risk assessment and therapeutic targets for recurrence-free treatment. There is mounting evidence of immune cell dysregulation in keloid scarring. In this study, we aimed to profile keloid scar tissues and blood cells and found that downregulation of cytotoxic CD8+ T cells is a keloid signature in the peripheral blood and keloid lesions. Single-cell RNA sequencing revealed that the NKG2A/CD94 complex was specifically upregulated, which might contribute to the significant reduction in CTLs within the scar tissue boundary. In addition, the NKG2A/CD94 complex was associated with high serum levels of soluble human leukocyte antigen-E (sHLA-E). We subsequently measured sHLA-E in our hospital-based study cohort, consisting of 104 keloid patients, 512 healthy donors, and 100 patients with an interfering disease. The sensitivity and specificity of sHLA-E were 83.69% (87/104) and 92.16% (564/612), respectively, and hypertrophic scars and other unrelated diseases exhibited minimal interference with the test results. Furthermore, intralesional therapy with triamcinolone combined with 5-fluorouracil drastically decreased the sHLA-E levels in keloid patients with better prognostic outcomes, while an incomplete reduction in the sHLA-E levels in patient serum was associated with higher recurrence. sHLA-E may effectively serve as a diagnostic marker for assessing the risk of keloid formation and a prognostic marker for the clinical outcomes of intralesional treatment.

[Retracted] Bergamottin isolated from Citrus bergamia exerts in vitro and in vivo antitumor activity in lung adenocarcinoma through the induction of apoptosis, cell cycle arrest, mitochondrial membrane potential loss and inhibition of cell migration and invasion.

Following the publication of the above paper, a concerned reader drew to the Editor's attention that a number of figures (specifically, Figs. 6, 8, 9, 10 and 12) contained apparent anomalies, including repeated patternings of data within the same figure panels. After having conducted an independent investigation in the Editorial Office, the Editor of Oncology Reports has determined that this paper should be retracted from the Journal on account of a lack of confidence concerning the originality and the authenticity of the data. The authors were asked for an explanation to account for these concerns, but the Editorial Office never received any reply. The Editor regrets any inconvenience that has been caused to the readership of the Journal. [the original article was published in Oncology Reports 36: 324­332, 2016; DOI: 10.3892/or.2016.4833].

High detection rates of colorectal neoplasia by stool DNA testing with a novel digital melt curve assay.

BACKGROUND & AIMS: Current stool DNA tests identify about half of individuals with colorectal cancers and miss most individuals with advanced adenomas. We developed a digital melt curve (DMC) assay to quantify low-abundance mutations in stool samples for detection of colorectal neoplasms and compared this test with other approaches. METHODS: We combined a melt curve assay with digital polymerase chain reaction and validated the quantitative range. We then evaluated its ability to detect neoplasms in 2 clinical studies. In study I, stool samples from patients with colorectal tumors with known mutations (KRAS, APC, BRAF, TP53) were assayed. In study II, archived stool samples from patients with advanced adenomas containing known KRAS mutations were assayed, along with controls. Results were compared with those from the stool DNA test PreGenPlus (Exact Sciences, Marlborough, MA), Hemoccult, and HemoccultSensa (both Beckman-Coulter, Fullerton, CA). RESULTS: The DMC assay detected samples in which only 0.1% of target genes were mutated. In study I, the DMC assay detected known mutations in 28 (90%) of 31 tumor samples and 6 (75%) of 8 advanced adenoma samples. In study II, the DMC assay detected 16 (59%) of 27 advanced adenoma samples that contained KRAS mutations, compared with 7% with the Hemoccult, 15% with the HemoccultSensa, and 26% with the PreGenPlus assays (P < .05 for each, compared with the DMC assay); specificities did not differ significantly. CONCLUSIONS: The DMC assay has a high level of sensitivity in detecting individuals with colon neoplasms and is better than current stool screening methods in detecting those with advanced adenomas. Further studies are indicated.

Robust performance of a novel stool DNA test of methylated SDC2 for colorectal cancer detection: a multicenter clinical study.

BACKGROUND AND AIMS: Stool DNA testing is an emerging and attractive option for colorectal cancer (CRC) screening. We previously evaluated the feasibility of a stool DNA (sDNA) test of methylated SDC2 for CRC detection. The aim of this study was to assess its performance in a multicenter clinical trial setting. METHODS: Each participant was required to undergo a sDNA test and a reference colonoscopy. The sDNA test consists of quantitative assessment of methylation status of SDC2 promoter. Results of real-time quantitative methylation-specific PCR were dichotomized as positive and negative, and the main evaluation indexes were sensitivity, specificity, and kappa value. All sDNA tests were performed and analyzed independently of colonoscopy. RESULTS: Among the 1110 participants from three clinical sites analyzed, 359 and 38 were diagnosed, respectively, with CRC and advanced adenomas by colonoscopy. The sensitivity of the sDNA test was 301/359 (83.8%) for CRC, 16/38 (42.1%) for advanced adenomas, and 134/154 (87.0%) for early stage CRC (stage I-II). Detection rate did not vary significantly according to age, tumor location, differentiation, and TNM stage, except for gender. The follow-up testing of 40 postoperative patients with CRC returned negative results as their tumors had been surgically removed. The specificity of the sDNA test was 699/713 (98.0%), and unrelated cancers and diseases did not seem to interfere with the testing. The kappa value was 0.84, implying an excellent diagnostic consistency between the sDNA test and colonoscopy. CONCLUSION: Noninvasive sDNA test using methylated SDC2 as the exclusive biomarker is a clinically viable and accurate CRC detection method. CHINESE CLINICAL TRIAL REGISTRY: Chi-CTR-TRC-1900026409, retrospectively registered on October 8, 2019; http://www.chictr.org.cn/edit.aspx?pid=43888&htm=4 .

Sulfatase 2 up-regulates glypican 3, promotes fibroblast growth factor signaling, and decreases survival in hepatocellular carcinoma.

UNLABELLED: It has been shown that the heparin-degrading endosulfatase, sulfatase 1 (SULF1), functions as a liver tumor suppressor, but the role of the related sulfatase, sulfatase 2 (SULF2), in liver carcinogenesis remains to be elucidated. We investigated the effect of SULF2 on liver tumorigenesis. Expression of SULF2 was increased in 79 (57%) of 139 hepatocellular carcinomas (HCCs) and 8 (73%) of 11 HCC cell lines. Forced expression of SULF2 increased HCC cell growth and migration, whereas knockdown of SULF2 using short hairpin RNA targeting SULF2 abrogated HCC cell proliferation and migration in vitro. Because SULF1 and SULF2 desulfate heparan sulfate proteoglycans (HSPGs) and the HSPG glypican 3 (GPC3) is up-regulated in HCC, we investigated the effects of SULF2 on GPC3 expression and the association of SULF2 with GPC3. SULF2-mediated cell growth was associated with increased binding of fibroblast growth factor 2 (FGF2), phosphorylation of extracellular signal-regulated kinase and AKT, and expression of GPC3. Knockdown of GPC3 attenuated FGF2 binding in SULF2-expressing HCC cells. The effects of SULF2 on up-regulation of GPC3 and tumor growth were confirmed in nude mouse xenografts. Moreover, HCC patients with increased SULF2 expression in resected HCC tissues had a worse prognosis and a higher rate of recurrence after surgery. CONCLUSION: In contrast to the tumor suppressor effect of SULF1, SULF2 has an oncogenic effect in HCC mediated in part through up-regulation of FGF signaling and GPC3 expression.

BMP3 suppresses colon tumorigenesis via ActRIIB/SMAD2-dependent and TAK1/JNK signaling pathways.

BACKGROUND: BMP3 gene is often found hypermethylated and hence inactivated in several types of cancers including colorectal cancer (CRC), indicating that it has a suppressor role in carcinogenesis. Though BMP3 is a reliable biomarker for screening CRC, the molecular mechanism of BMP3 in carcinogenesis remains largely unknown. METHODS: The expression level of BMP3 was examined by immunohistochemistry staining and western blot. Methylation-specific PCR (MSP) and real-time quantitative MSP were used to test the hypermethylation status of BMP3 gene. Analyses of BMP3 function in colon cancer cell proliferation, migration, invasion, and apoptosis were performed using HCT116 and KM12 cells. BMP3 was further knocked down or overexpressed in CRC cells, and the effects on cell growth of xenograft tumors in nude mice were assessed. Co-immunoprecipitation and immunofluorescence staining were used to analyze the association between BMP3 and BMPR2 or BMP3 and ActRIIB. Microarray analysis was performed to identify most differentially expressed genes and pathways regulated by BMP3. The BMP3-regulated SMAD2-dependent signaling pathway and TAK1/JNK signal axes were further investigated by quantitative PCR and western blot. RESULTS: BMP3 gene was hypermethylated and its expression was downregulated in both CRC tissues and cell lines. Expressing exogenous BMP3 in HCT116 inhibited cell growth, migration, and invasion and increased rate of apoptosis both in vitro and in vivo. However, shRNA-mediated attenuation of endogenous BMP3 in KM12 reversed such inhibitory and apoptotic effects. Furthermore, BMP3 could bind to ActRIIB, an activin type II receptor at the cellular membrane, thereby activating SMAD2-dependent pathway and TAK1/JNK signal axes to regulate downstream targets including caspase-7, p21, and SMAD4 that play crucial roles in cell cycle control and apoptosis. CONCLUSIONS: Our study reveals a previously unknown mechanism of BMP3 tumor suppression in CRC and provides a rationale for future investigation of BMP3 as a potential target for the development of novel therapeutic agents to fight CRC.

Secreted frizzled-related protein 1 loss contributes to tumor phenotype of clear cell renal cell carcinoma.

PURPOSE: Incidence and mortality rates for renal cell carcinoma (RCC) have been rising for decades. Unfortunately, the molecular events that support RCC carcinogenesis remain poorly understood. In an effort to gain a better understanding of signaling events in clear cell RCC (cRCC), we investigated the antitumor activity of secreted frizzled-related protein 1 (sFRP1), a negative regulator of Wnt signaling. EXPERIMENTAL DESIGN: Genomic profiling of cRCC tumors and patient-matched normal tissues was done and confirmed using quantitative PCR and immunohistochemistry. Methylation-specific PCR was done on patient samples to evaluate the mechanism responsible for sFRP1 loss. sFRP1 expression was restored in cRCC cells and the effects on tumor phenotype were characterized. RESULTS: Genomic profiling, quantitative PCR, and immunohistochemistry indicated that loss of sFRP1 occurred in cRCC and papillary RCC patient tissues. Twelve Wnt-regulated genes were up-regulated in cRCC tissues, including c-myc and cyclin D1, potentiators of cell proliferation and survival. Methylation of the sFRP1 gene was one mechanism identified for attenuation of sFRP1 mRNA. Stable reexpression of sFRP1 in cRCC cells resulted in decreased expression of Wnt target genes, decreased growth in cell culture, inhibition of anchorage-independent growth, and decreased tumor growth in athymic nude mice. CONCLUSIONS: To our knowledge, this is the first report to show that stable restoration of sFRP1 expression in cRCC cells attenuates the cRCC tumor phenotype. Our data support a role for sFRP1 as a tumor suppressor in cRCC and that perhaps loss of sFRP1 is an early, aberrant molecular event in renal cell carcinogenesis.

Highly methylated genes in colorectal neoplasia: implications for screening.

Discriminant markers are required for accurate cancer screening. We evaluated genes frequently methylated in colorectal neoplasia to identify the most discriminant ones. Four genes specifically methylated in colorectal cancer [bone morphogenetic protein 3 (BMP3), EYA2, aristaless-like homeobox-4 (ALX4), and vimentin] were selected from 41 candidate genes and evaluated on 74 cancers, 62 adenomas, and 70 normal epithelia. Methylation status was analyzed qualitatively and quantitatively and confirmed by bisulfite genomic sequencing. Effect of methylation on gene expression was evaluated in five colon cancer cell lines. K-ras and BRAF mutations were detected by sequencing. Methylation of BMP3, EYA2, ALX4, or vimentin was detected respectively in 66%, 66%, 68%, and 72% of cancers; 74%, 48%, 89%, and 84% of adenomas; and 7%, 5%, 11%, and 11% of normal epithelia (P < 0.01, cancer or adenoma versus normal). Based on area under the curve analyses, discrimination was not significantly improved by combining markers. Comethylation was frequent (two genes or more in 72% of cancers and 84% of adenomas), associated with proximal neoplasm site (P < 0.001), and linked with both BRAF and K-ras mutations (P < 0.01). Cell line experiments supported silencing of expression by methylation in all four study genes. This study shows BMP3, EYA2, ALX4, and vimentin genes are methylated in most colorectal neoplasms but rarely in normal epithelia. Comethylation of these genes is common, and pursuit of complementary markers for methylation-negative neoplasms is a rational strategy to optimize screening sensitivity.

Detection of colorectal disease by stool defensin assay: an exploratory study.

BACKGROUND & AIMS: Alpha-defensins 1-3 (human neutrophil peptides [HNP]1-3), reported to be elevated in tumor tissue and serum of patients with colorectal cancer (CRC), have not been studied in stool. We evaluated the neoplasm specificity of HPN1-3 and their discriminant value as stool markers for CRC. METHODS: Protein and mRNA expression of HPN1-3 were assayed in CRC cell lines, microdissected CRC and normal epithelium, and white blood cells. HNP1-3 proteins in stools were quantified in blinded fashion from 30 normal subjects, 20 patients with CRC, 10 with a large colorectal adenoma, 10 with upper gastrointestinal cancer, and 10 with IBD. Stool lactoferrin was also quantified. RESULTS: HPN1-3 proteins were not detected in CRC cell lines but were high (>4000 ng/mL) in white blood cells. mRNA levels of HPN1-3 were comparably low in CRC cell lines, microdissected CRC, and normal colon epithelium, but they were >1000-fold and >30,000-fold higher in white blood cells and neutrophils, respectively. Mean stool HPN1-3 levels were 17 ng/mL with normals, 125 ng/mL with CRC, 62 ng/mL with adenoma, 63 ng/mL with upper gastrointestinal cancer, and 231 ng/mL with IBD (P < .01 for each patient group vs normals). HPN1-3 levels in IBD were higher than in CRC (P = .04). At 90% specificity, sensitivity of stool defensins was 35% for CRC, 40% for adenoma, 40% for upper gastrointestinal cancers, and 80% for IBD. Stool defensins and lactoferrin levels correlated (R2 = 0.70, P < .001). CONCLUSIONS: Alpha-defensins 1-3 levels are nonspecifically elevated in stools from patients with colorectal neoplasia and likely originate from white blood cells. Alpha-defensins 1-3 in stool might serve as markers of inflammatory bowel conditions.

FAM83D knockdown regulates proliferation, migration and invasion of colorectal cancer through inhibiting FBXW7/Notch-1 signalling pathway.

Colorectal cancer is one of the most common malignant tumors in the digestive system, and in China its incidence is rising in recent years. The FAM83D family with sequence similarity 83, member D is associated with the occurrence and development of various cancers. However, in human colorectal cancer, the biological function of FAM83D and its molecular mechanism are still little known. In our study, we found that FAM83D mRNA expression level was markedly up-regulated in colorectal cancerous tissues when compared with that of adjacent normal colon tissues. We also found that the protein and mRNA expression levels of FAM83D are dramatically increased in human colorectal cancer cell lines, including Caco-2, RKO, DLD-1, HT-29, LoVo, SW480, and HCT116, especially in SW480 and HCT116 cells, when compared with that of human normal colon epithelial cell line (NCM460). Next, in HCT116 and SW480 cells, the biological function of FAM83D was examined. FAM83D-knockdown notably inhibited cell proliferation and colony formation. Cell apoptosis was promoted by FAM83D knockdown. In addition, FAM83D siRNA decreased cell migration and invasion. Moreover, FAM83D knockdown up-regulated the protein expression level of F-box and WD repeat domain-containing 7 (FBXW7), but diminished the Notch1 protein expression level. It also found that FBXW7 siRNA reverses the suppressive effect of FAM83D knockdown on Notch1 protein expression. Notch1 overexpression reversed the effect of FAM83D knockdown on colorectal cancer cell proliferation, cell migration and invasion. In conclusion, FAM83D knockdown promoted colorectal cancer cell apoptosis, inhibited cell proliferation, cell migration and invasion, which might be associated with inhibiting the FBXW7/Notch1 signal pathway. Our findings indicated that FAM83D is a promising molecular target for colorectal cancer treatment.

Stool DNA Test of Methylated Syndecan-2 for the Early Detection of Colorectal Neoplasia.

Background: Although the incidence of colorectal cancer is steadily increasing, screening for colorectal cancer with conventional approaches is not routinely performed in China. Noninvasive screening methods are attractive options to resolve this issue. Syndecan-2 (SDC2) is frequently methylated in colorectal cancer. However, the value of a stool test of methylated SDC2 for the detection of colorectal cancer is unknown.Methods: Methylation status of SDC2 was tested in cell lines and 398 colorectal tissue samples and further evaluated with 497 stool samples, including 196 from colorectal cancer patients, 122 from adenoma patients, and 179 from normal individuals, using real-time methylation-specific PCR. The impacts of one quantitative partial stool sampling device and 17 potentially interfering substances on the performance of fecal methylated SDC2 were also analyzed. SDC2 expression was also measured.Results:SDC2 methylation level was higher in 96.8% (120/124) of colorectal cancer tissues compared with paired adjacent normal epithelia. Stool test of methylated SDC2 detected 81.1% (159/196) of colorectal cancer and 58.2% (71/122) of adenomas at a specificity of 93.3% (167/179). No significant difference was found between partial and whole stool collection on colorectal cancer detection (P > 0.05, R2 = 0.80). Among 17 interfering substances, only berberine at high concentrations inhibited fecal detection of methylated SDC2SDC2 was overexpressed in colorectal cancer tissues compared with normal epithelia.Conclusions: Fecal methylated SDC2 is a valuable biomarker for the noninvasive detection of colorectal neoplasms.Impact: Stool DNA test of methylated SDC2 would serve as an alternative method for screening colorectal neoplasms. Cancer Epidemiol Biomarkers Prev; 26(9); 1411-9. ©2017 AACR.

Overexpression of MSK1 is associated with tumor aggressiveness and poor prognosis in colorectal cancer.

BACKGROUND/AIMS: Mitogen- and stress-activated protein kinase 1 (MSK1) has recently been implicated in cell proliferation and neoplastic transformation. However, the involvement of MSK1 in colorectal cancer (CRC) has not been addressed. This study aimed to evaluate the expression and potential functions of MSK1 in CRC. METHODS: The MSK1 expression was investigated by immunohistochemistry, western blot and reverse transcription-polymerase chain reaction. The associations between clinicopathological characteristics and MSK1 expression were assessed. Kaplan-Meier analysis and Cox regression models were carried out. CRC cells with MSK1 knockdown or overexpression were generated. A range of experiments were performed to demonstrate MSK1's role in CRC. RESULTS: MSK1 was overexpressed in 148 out of 329 CRC patients. CRC patients with high MSK1 expression had shorter overall survival than those with low MSK1 (P=0.033), especially among patients with stage III tumors (P=0.005). Knockdown of MSK1 in CRC cells suppressed cell proliferation, anchorage-independent growth, migration and invasion, and promoted 5-fluorouracil chemosensitivity and intracellular NADP+/NADPH ratio. However, overexpression of MSK1 had the opposite effects. CONCLUSIONS: Overexpression of MSK1 is associated with poor prognosis in CRC and is connected to tumor aggressiveness. MSK1 is a potential target for new therapies and a candidate of biomarker for prognosis.