Everolimus exerts anticancer effects through inhibiting the interaction of matrix metalloproteinase-7 with syndecan-2 in colon cancer cells.

Previous work showed that matrix metalloproteinase-7 (MMP-7) regulates colon cancer activities through an interaction with syndecan-2 (SDC-2) and SDC-2-derived peptide that disrupts this interaction and exhibits anticancer activity in colon cancer. Here, to identify potential anticancer agents, a library of 1,379 Food and Drug Administration (FDA)-approved drugs that interact with the MMP-7 prodomain were virtually screened by protein-ligand docking score analysis using the GalaxyDock3 program. Among five candidates selected based on their structures and total energy values for interacting with the MMP-7 prodomain, the known mechanistic target of rapamycin kinase (mTOR) inhibitor, everolimus, showed the highest binding affinity and the strongest ability to disrupt the interaction of the MMP-7 prodomain with the SDC-2 extracellular domain in vitro. Everolimus treatment of the HCT116 human colon cancer cell line did not affect the mRNA expression levels of MMP-7 and SDC-2 but reduced the adhesion of cells to MMP-7 prodomain-coated plates and the cell-surface localization of MMP-7. Thus, everolimus appears to inhibit the interaction between MMP-7 and SDC-2. Everolimus treatment of HCT116 cells also reduced their gelatin-degradation activity and anticancer activities, including colony formation. Interestingly, cells treated with sirolimus, another mTOR inhibitor, triggered less gelatin-degradation activity, suggesting that this inhibitory effect of everolimus was not due to inhibition of the mTOR pathway. Consistently, everolimus inhibited the colony-forming ability of mTOR-resistant HT29 cells. Together, these data suggest that, in addition to inhibiting mTOR signaling, everolimus exerts anticancer activity by interfering with the interaction of MMP-7 and SDC-2, and could be a useful therapeutic anticancer drug for colon cancer.NEW & NOTEWORTHY The utility of cancer therapeutics targeting the proteolytic activities of MMPs is limited because MMPs are widely distributed throughout the body and involved in many different aspects of cell functions. This work specifically targets the activation of MMP-7 through its interaction with syndecan-2. Notably, everolimus, a known mTOR inhibitor, blocked this interaction, demonstrating a novel role for everolimus in inhibiting mTOR signaling and impairing the interaction of MMP-7 with syndecan-2 in colon cancer.

Significance of SDC2 and NDRG4 methylation in stool for colorectal cancer diagnosis.

BACKGROUND: Recent studies have identified methylated SDC2 and NDRG4 in colorectal cancer (CRC), however, the diagnostic value of the combined two genes remains undefined. This study aims to investigate the methylation of SDC2 and NDRG4 in stool samples and their application in diagnosis of CRC. METHODS: Five groups were enrolled in our study which consisted of CRC (n = 138), advanced adenomas (n = 27), polyp (n = 35), intestinal disease control (n = 150), and healthy individuals (n = 28). Methylation status of SDC2 and NDRG4 in fecal samples were tested with appropriate commercial kits. Primary data were collected and statistical analyses were performed. RESULTS: The positive rates of both SDC2 and NDRG4 methylation in stool samples of CRC group were significantly higher (P < 0.001) than those of either group of advanced adenomas, or polyp, or intestinal disease or the healthy control. It was suggested that both methylated SDC2,NDRG4, SDC2/NDRG4 and age were independent risk factors for CRC. The sensitivity of SDC2 and NDRG4 for CRC diagnosis were 73.9 % and 63.0 %, respectively, while SDC2 combined with NDRG4 had a higher sensitivity of 85.5 %. The specificity of SDC2, NDRG4 and SDC2 combined with NDRG4 achieved 91.6 %, 88.3 % and 84.6 %, respectively. The AUC for methylated SDC2 and NDRG4 were 0.828 (95 % CI: 0.780-0.876) and 0.757 (95 % CI: 0.703-0.811), respectively. In contrast, SDC2 combined with NDRG4 improved the AUC to 0.850 (95 % CI: 0.807-0.893). CONCLUSIONS: This research confirmed the significance of detection of SDC2 and NDRG4 methylation by using noninvasive samples of stool. More importantly, attributing to their high level and frequency of methylation in stool, SDC2 and NDRG4 could be promising biomarkers for stool-based method for screening and early diagnosis of CRC, especially when combined.

Evaluating the clinical performance of SDC2/NDRG4 methylation for colorectal cancer detection.

Purpose: The performance and clinical accuracy of combined SDC2/NDRG4 methylation were evaluated in diagnosing colorectal cancer (CRC) and advanced adenoma. Methods: A total of 2333 participants were enrolled to assess the sensitivity and specificity of biomarkers in diagnosing CRC in a multicenter clinical trial through feces DNA methylation tests. Results: SDC2/NDRG4 methylation showed excellent performance for CRC detection in biomarker research and the real world. Its sensitivity for detecting CRC, early CRC and advanced adenoma were 92.06%, 91.45% and 62.61%, respectively. Its specificity was 94.29%, with a total coincidence rate of 88.28%. When interference samples were included, the specificity was still good (82.61%). Therefore, the SDC2/NDRG4 methylation test showed excellent performance in detecting CRC and advanced adenoma under clinical application.

Colorectal cancer (CRC) is one of the most malignant tumors of the digestive system and second only to breast cancer and lung cancer in terms of global incidence. Early CRCs are challenging to determine given their atypical nature. In contrast, late CRC symptoms are affected by the type, location and range of the lesion and complications. Therefore, CRC patients are generally diagnosed late, present with a high degree of malignancy, and have poor prognosis and 5-year survival rates. The current study therefore evaluated whether SDC2 and NDRG4 methylation could be used for diagnosis CRCs at an early stage and whether it has the potential to detect asymptomatic patients with adenomas. The findings presented herein will certainly help support the early diagnosis of CRC and precancerous lesions in clinical practice.

Value of faecal exfoliated cells in colorectal tumour screening using SDC2 methylation test.

BACKGROUND: This study aimed to evaluate the diagnostic value of a non-invasive methylation gene test in clinical colorectal tumour screening. METHOD: The quantitative methylation-specific PCR technique was used to detect faecal methylated syndecan-2 (mSDC2) in patients who received the screening of colorectal cancer (CRC).To evaluate the positive predictive value (PPV) of mSDC2 in patients with colorectal cancer, advanced adenoma (AA), and colorectal tumor (CRN) in risk factor stratification. RESULTS: The PPV of CRC, CRC + AA and CRN in male patients were 28.03%, 43.55% and 56.24%, respectively, which were higher than female patients. The positive detection rate of mSDC2 and the PPV of CRC gradually increased with age; The PPV in patients aged over 80 years was up to 78.05%, which was more significant than in younger patients with CRC. The PPV of CRC, AA and CRN were 37.10%, 11.80% and 63.37%, respectively. mSDC2 has a high detection rate of 85-100% in AA with intramucosal carcinoma alone or in combination with severe atypical hyperplasia or villous adenoma. CONCLUSION: The mSDC2 test has a higher PPV in patients with colorectal cancer and colorectal adenoma (AD), especially in high-risk groups over 50 years of age, and may help in the early diagnosis of colorectal tumours in the future.

Correlation between Stool DNA-based SDC2 Methylation Tests Combined with Tumor Markers with Pathological Features in Colorectal Cancer.

BACKGROUND: Colorectal cancer (CRC) screening is the most efficient strategy to reduce disease-related mortality. In this study, we aimed to investigate the association of a methylation-based stool DNA test with serum protein biomarker panel (CEA, CA125, CA199, and AFP) in CSC patients and their relationship with pathological features to improve the diagnostic efficacy and applicability in CSC in the Chinese population. METHODS: In this double-blinded case-control study, we enrolled 150 participants from our hospital, including 50 CRC patients, 50 adenomas, and 50 healthy controls. We compared the cycling threshold (Ct) values of stool DNA-based SDC2 measured by quantitative methylation-specific PCR (MSP) in the three groups. We also evaluated the differences and correlation between serum concentrations of tumor biomarker and pathological features in patients with CSC, including TNM stage (I, II, III), tumor size, and lymph node metastasis. The discrimination performance of indexes was assessed using sensitivity, specificity, and the area under the receiver operating characteristic curve (AUC). RESULTS: CSC was more common in middle-aged people and men. The methylation-based stool DNA test was not significantly correlated with other tumor indicators except CEA, and the difference was statistically significant. Compared with the normal control group, the combined diagnostic value of the methylation-based stool DNA test and tumor indicators was significantly higher than individual biomarkers alone, especially the methylation-based stool DNA test combined with CEA and AFP, which improved the AUC to 0.96. This combination can increase the positive rate of pathological stage diagnosis. CONCLUSIONS: Combining a methylation-based stool DNA test with CEA and AFP can significantly improve the diagnostic value of CRC and can be used to confirm the diagnosis of colorectal cancer. This combination can also be used as a reliable indicator identifying early-stage CRC patients and pathology. A large-scale study is underway to further define the clinical application of this method for the diagnosis of CRC among Chinese populations.

Combined detection of SDC2/ADHFE1/PPP2R5C methylation in stool DNA for colorectal cancer screening.

BACKGROUND: Colorectal cancer (CRC) is a disease of global concern, and its increasing incidence suggests the need for early and accurate diagnosis. The aim of this study was to investigate the value of combined detection of SDC2, ADHFE1 and PPP2R5C gene methylation in stool samples for early CRC screening. METHODS: Stool samples from patients with CRC (n = 105), advanced adenoma (AA) (n = 54), non-advanced adenoma (NA) (n = 57), hyperplastic or other polyps (HOP) (n = 47) or no evidence of disease (NED) (n = 100) were collected from September 2021 to September 2022. The methylation levels of SDC2, ADHFE1 and PPP2R5C were quantified by quantitative methylation-specific polymerase chain reaction (qMSP), and faecal immunochemical testing (FIT) was performed. The diagnostic value was assessed using reporter operating characteristic (ROC) curve analysis. RESULTS: The sensitivity of combined detection of SDC2/ADHFE1/PPP2R5C methylation in predicting CRC (0-IV) was 84.8%, the specificity was 98.0%, and the AUC was 0.930 (95% CI 0.889-0.970). Compared to FIT and serum tumour biomarkers, it showed better diagnostic performance for different stages of CRC. CONCLUSION: The results of this study verified that the methylation levels of SDC2, ADHFE1 and PPP2R5C in stool DNA were significantly increased in CRC patients. Combined detection of SDC2/ADHFE1/PPP2R5C methylation is a potential non-invasive diagnostic method for CRC and precancerous lesion screening. CLINICAL TRIAL REGISTRATION: Chinese Clinical Trials Registry, ChiCTR2100046662, registered on 26 May 2021, prospective registration.

[Methylated SDC2 testing in stool DNA for early screening of colorectal cancer in Shipai Town, Dongguan City].

Objective: To explore the utility of stool-based DNA test of methylated SDC2 (mSDC2) for colorectal cancer (CRC) screening in residents of Shipai Town, Dongguan City. Methods: This was a cross-sectional study. Using a cluster sampling method, residents of 18 villages in Shipai Town, Dongguan City were screened for CRC from May 2021 to February 2022. In this study, mSDC2 testing was employed as a preliminary screening method. Colonoscopy examination was recommended for individuals identified as high-risk based on the positive mSDC2 tests. The final screening results, including the rate of positive mSDC2 tests, the rate of colonoscopy compliance, the rate of lesions detection, and the cost-effectiveness of screening, were analyzed to explore the benefits of this screening strategy. Results: A total of 10 708 residents were enrolled and completed mSDC2 testing, giving a participation rate of 54.99% (10 708/19 474) and a pass rate of 97.87% (10 708/10 941). These individuals included 4 713 men (44.01%) and 5 995 women (55.99%) with a mean age of (54.52±9.64) years. The participants were allocated to four age groups (40-49, 50-59, 60-69, and 70-74 years), comprising 35.21%(3770/10 708), 36.25% (3882/10 708), 18.84% (2017/10 708), and 9.70% (1039/10 708) of all participants, respectively. mSDC2 testing was positive in 821/10 708 (7.67%) participants, 521 of whom underwent colonoscopy, resulting in a compliance rate of 63.46% (521/821). After eliminating of 8 individuals without pathology results, data from 513 individuals were finally analyzed. Colonoscopy detection rate differed significantly between age groups (χ2=23.155, P<0.001),ranging from a low of 60.74% in the 40-49 year age group to a high of 86.11% in the 70-74 year age group. Colonoscopies resulted in the diagnosis of 25 (4.87%) CRCs, 192 (37.43%) advanced adenomas, 67 (13.06%) early adenomas, 15 (2.92%) serrated polyps, and 86 (16.76%) non- adenomatous polyps. The 25 CRCs were Stage 0 in 14 (56.0%) individuals, stage I in 4 (16.0%), and Stage II in 7(28.0%). Thus, 18 of the detected CRCs were at an early stage. The early detection rate of CRCs and advanced adenomas was 96.77% (210/217). The rate of mSDC2 testing for all intestinal lesions was 75.05% (385/513). In particular, the financial benefit of this screening was 32.64 million yuan, and the benefit-cost ratio was 6.0. Conclusion: Screening for CRCs using stool-based mSDC2 testing combined with colonoscopy has a high lesion detection rate and a high cost-effectiveness ratio. This is a CRC screening strategy that deserves to be promoted in China.

Methylated SDC2 testing in stool DNA for early screening of colorectal cancer in Shipai Town, Dongguan City / 中华胃肠外科杂志

Objective: To explore the utility of stool-based DNA test of methylated SDC2 (mSDC2) for colorectal cancer (CRC) screening in residents of Shipai Town, Dongguan City. Methods: This was a cross-sectional study. Using a cluster sampling method, residents of 18 villages in Shipai Town, Dongguan City were screened for CRC from May 2021 to February 2022. In this study, mSDC2 testing was employed as a preliminary screening method. Colonoscopy examination was recommended for individuals identified as high-risk based on the positive mSDC2 tests. The final screening results, including the rate of positive mSDC2 tests, the rate of colonoscopy compliance, the rate of lesions detection, and the cost-effectiveness of screening, were analyzed to explore the benefits of this screening strategy. Results: A total of 10 708 residents were enrolled and completed mSDC2 testing, giving a participation rate of 54.99% (10 708/19 474) and a pass rate of 97.87% (10 708/10 941). These individuals included 4 713 men (44.01%) and 5 995 women (55.99%) with a mean age of (54.52±9.64) years. The participants were allocated to four age groups (40-49, 50-59, 60-69, and 70-74 years), comprising 35.21%(3770/10 708), 36.25% (3882/10 708), 18.84% (2017/10 708), and 9.70% (1039/10 708) of all participants, respectively. mSDC2 testing was positive in 821/10 708 (7.67%) participants, 521 of whom underwent colonoscopy, resulting in a compliance rate of 63.46% (521/821). After eliminating of 8 individuals without pathology results, data from 513 individuals were finally analyzed. Colonoscopy detection rate differed significantly between age groups (χ2=23.155, P<0.001),ranging from a low of 60.74% in the 40-49 year age group to a high of 86.11% in the 70-74 year age group. Colonoscopies resulted in the diagnosis of 25 (4.87%) CRCs, 192 (37.43%) advanced adenomas, 67 (13.06%) early adenomas, 15 (2.92%) serrated polyps, and 86 (16.76%) non- adenomatous polyps. The 25 CRCs were Stage 0 in 14 (56.0%) individuals, stage I in 4 (16.0%), and Stage II in 7(28.0%). Thus, 18 of the detected CRCs were at an early stage. The early detection rate of CRCs and advanced adenomas was 96.77% (210/217). The rate of mSDC2 testing for all intestinal lesions was 75.05% (385/513). In particular, the financial benefit of this screening was 32.64 million yuan, and the benefit-cost ratio was 6.0. Conclusion: Screening for CRCs using stool-based mSDC2 testing combined with colonoscopy has a high lesion detection rate and a high cost-effectiveness ratio. This is a CRC screening strategy that deserves to be promoted in China.

[Application study of stool-based methylated SDC2 test in the screening of colorectal neoplasms for physical examination population].

Objective: To investigate the value of stool-based methylated SDC2 test in physical examination population for the screening of colorectal neoplasms. Methods: Using the prospective cohort study method, from December 2020 to November 2021, 2 107 participants from the First People's Hospital of Xiushui County, Jiangxi Province were enrolled, consisted of 1 012 males and 1 094 females, aged 20-90 years with the median age of 49 years old. Fresh stool samples were collected and SDC2 DNA methylation tests were carried out as the primary screening method. The participants with positive results were recommended to undergo colonoscopy, and those who were negative were followed up by telephone. The positive rate of screening, the compliance of colonoscopy, and the detection of colorectal lesions were analyzed by chi-square test. Combined the follow-up results of negative subjects, the value of SDC2 DNA methylation test for the screening of colorectal neoplasms was evaluated. Results: Among the 2 107 participants, 2 106 completed the SDC2 methylation test. 113 participants (5.4%) were positive. The positive rate of primary screening increased with age significantly (χ2=32.135, P<0.001). Out of 113 cases, 72 (63.7%) underwent colonoscopy examinations. Finally, 3 (4.2%) cases of colorectal cancer, 12 (16.7%) cases of advanced adenoma, 31 (43.1%) cases of non-advanced adenoma, and 16 (22.2%) cases of non-adenomatous polyp were detected. The positive predictive value (PPV) of stool-based SDC2 DNA methylation test for intestinal lesions and colorectal neoplasms were 86.1% and 63.9%, respectively. Among the 1 374 follow-up participants, the negative predictive value (NPV) of this test for intestinal lesions and colorectal neoplasms were 97.7% and 99.4%, respectively. Conclusion: Primary stool-based SDC2 DNA methylation test and subsequent colonoscopy examination can effectively find colorectal neoplasms. This strategy may be a potential tool for the screening of colorectal neoplasms in general risk population.

Analysis of SDC2 gene promoter methylation in whole blood for noninvasive early detection of colorectal cancer.

Objectives: Considering the limitations of the current approaches to colorectal cancer (CRC) screening, scientists strived to find noninvasive and more powerful biomarkers for the early diagnosis of CRC. Nowadays, there are different sources of biomarkers for CRC diagnosis. Blood-based samples including circulating cell-free tumor DNA (ctDNA) and DNA extracted from leukocytes in peripheral blood might be promising sources of noninvasive cancer biomarkers such as cancer-specific methylation patterns. In this study, we aimed to evaluate the noninvasive early diagnosis of CRC via quantitative promotor methylation analysis of SDC2 gene in whole blood. Materials and Methods: Sixty-five CRC patients and 65 healthy participants were enrolled to assess promoter methylation of SDC2 gene in whole blood using the methylation quantification endonuclease-resistant DNA (MethyQESD) technique. Results: Our findings demonstrated drastic hypermethylation of SDC2 in blood samples from CRC subjects (37.91%) compared with non-malignant individuals (17.02%) (P < 0.001). The sensitivity for detection of CRC by methylation of SDC2 was 81.54%, with a specificity of 69.23%. The ROC curve analysis demonstrated that the AUC was 0.847 (P < 0.001), indicating that the status of SDC2 promoter methylation in whole blood is an excellent biomarker of CRC diagnosis. Furthermore, our results showed that methylation level in CRC patients significantly increased in higher tumor stages, demonstrating that an increased percentage of methylation is correlated with tumor progression (P < 0.001). Conclusion: SDC2 promoter methylation status in blood samples is a valuable cancer biomarker and holds high power and accuracy in distinguishing CRC patients from healthy subjects in the early stages of the disease.

A simplified multiplex methylated DNA testing for early detection of colorectal cancer in stool DNA.

BACKGROUND: ColoDefense1.0 assay has demonstrated its excellent sensitivity and specificity for early detection of colorectal cancer (CRC) by detecting the methylation levels of SDC2 and SEPT9, while exhibited limitations on relatively large sample capacity required and limited detection throughput by applying triplicate PCR reactions for each sample. In this study, ColoDefense1.0 was simplified and optimized into ColoDefense2.0 in a single PCR reaction. METHODS: A total 529 stool specimens were collected, and 244 CRC patients, 34 patients with advanced adenomas (AA), 64 with small polyps (SP) and 187 control subjects were divided in training and validation cohorts. Methylation levels of SEPT9 and SDC2 were examined by qPCR reactions in triplicate or single. RESULTS: The stool DNA quantity stored in preservative buffer at 37 °C up to 7 days exhibited no significant decrease. In the training cohort, when the number of replicates reduced from 3 to 1, the overall performance of ColoDefense2.0 was identical to that of ColoDefense1.0, showing sensitivities of 71.4% for AA and 90.8% for all stage CRC with a specificity of 92.9%. In the validation cohort, sensitivities of SP, AA and CRC using ColoDefense2.0 were 25.0%, 55.0% and 88.2%, increased from 14.1% (20.3%), 40.0% (40.0%) and 79.4% (67.6%) using SDC2 (SEPT9) alone; along with an overall specificity of 90.2%, decreased from 94.1% (95.1%) using SDC2 (SEPT9) alone. CONCLUSION: The simplified ColoDefense test maintained the overall performance while reduced the number of PCR reactions to 1/3, and provided an effective and convenient tool to detect early CRC and precancerous lesions and potentially improve the compliance of screening.

Diagnostic accuracy of DNA-based SDC2 methylation test in colorectal cancer screening: a meta-analysis.

BACKGROUND: A growing body of research suggests that methylated genes can be used as early diagnostic markers for cancer. Some studies on methylated Syndecan 2 (SDC2) have shown that it has a great diagnostic ability in colorectal cancer. This meta-analysis was aimed to estimate the diagnostic performance of methylated SDC2 as a potential novel biomarker to screen for the colorectal cancer. METHODS: Two independent researchers conducted a comprehensive literature search to identify all relevant studies on SDC2 methylation for the diagnosis of colorectal cancer from inception to March 1, 2021. By using STATA and Revman software, the data were analyzed using a Bivariate mixed model. The quality of each study was also evaluated. RESULTS: A total of 12 studies comprised of 1574 colorectal cancer patients and 1945 healthy people were included in our meta-analysis. Bivariate analysis showed a pooled sensitivity of 0.81 [95% confidence interval (CI) 0.74-0.86], specificity of 0.95 (95% CI 0.93-0.96), positive likelihood ratio of 15.29 (95% CI 10.83-21.60), and negative likelihood ratio of 0.21 (95% CI 0.15-0.27). The diagnostic odds ratio and the area under the summary ROC curve for diagnosing colorectal cancer were 74.42 (95% CI45.44-121.89) and 0.96 (95% CI 0.94-0.97), respectively. For adenomas, the pooled sensitivity and specificity were 0.47 (95% CI 0.34-0.61) and 0.95 (95% CI 0.92-0.97), respectively. CONCLUSIONS: Our analysis revealed that methylated SDC2 could be considered as a potential novel biomarker to screen for colorectal cancer.

Evaluation of combined detection of multigene mutation and SDC2/SFRP2 methylation in stool specimens for colorectal cancer early diagnosis.

PURPOSE: Molecular diagnostics of colorectal cancer (CRC) can be used as an auxiliary approach for patients recommended for colonoscopy, providing more CRC supplemental diagnosis options. This study investigated whether combined detection of KRAS/BRAF/APC mutation and SDC2/SFRP2 methylation can serve as auxiliary diagnostics in clinical management. METHODS: KRAS/BRAF/APC mutation and SDC2/SFRP2 methylation in stool samples from healthy donors, patients with CRC, advanced adenoma (AA), non-advanced adenoma (NAA), or other gastroenterological diseases were evaluated using quantitative PCR (qPCR) or methylation-specific quantitative PCR (MSP). Test accuracy was determined by evaluating the tests' sensitivity, specificity, positive/negative predictive value (PPV/NPV), or positive/negative likelihood ratio (PLR/NLR). RESULTS: The combined fecal KRAS/BRAF/APC mutation and SFRP2/SDC2 methylation detection test achieved a sensitivity of 88.57% with a PPV of 93.64% and a PLR of 7.10 for CRC patients. In comparison, the corresponding parameters for multigene mutation were 46.67%, 92.59%, and 36.26 and 83.81%, 93.94%, and 7.47, for DNA methylation, separately. The sensitivity of the combined test, gene mutation test, and DNA methylation test approach was 75%, 28.26%, and 72.83%. Furthermore, the specificity of this approach in the NAA group was 79.49%. Meanwhile, the overall diagnostic specificity for the combined test in NAA, healthy control, and interference groups was 88.42%. In addition, the sensitivity of the combined detection method increased with the disease stage in CRC patients and elevated along with the lesion size (≥ 1 cm) in AA patients. CONCLUSION: Combined detection of fecal KRAS/BRAF/APC mutation and SFRP2/SDC2 methylation has potential application value for the auxiliary diagnosis of CRC and AA.

A novel method for early detection of colorectal cancer based on detection of methylation of two fragments of syndecan-2 (SDC2) in stool DNA.

BACKGROUND: Methylated SDC2 has been proved as a diagnostic marker for human colorectal cancer (CRC), noninvasive stool DNA-based methylation testing also emerges as a novel approach for detecting CRC. The aim of this study was to evaluate the clinical performance of stool DNA-based SDC2 methylation test by a new qPCR detection reagent for early detection of CRC. METHODS: A new qPCR detection reagent contained two differentially methylated regions in SDC2 CpG islands for the detection of CRC was used in this study. Performance of the SDC2 methylation detection reagent was evaluated by analyzing limit of detection, precision, and specificity. The effect of interfering substances on assay performance was also tested. 339 subjects (102 CRC patients, 50 patients with advanced adenomas, 39 patients with non-advanced adenomas, 18 colitis patients and 130 normal individuals) from the China-Japan Friendship Hospital were evaluated. Approximately 2.5 g of stool sample was collected from each participant. Stool DNA was extracted and bisulfite-converted, followed by qPCR assay, which contained two pairs of primers for the methylation detection of two fragments of the SDC2 gene (named SDC2-A and SDC2-B). The diagnostic value of this test in CRC was evaluated by calculating receiver operating characteristic (ROC) curve, and value of the area under the curve (AUC). RESULTS: The test kit was able to detect methylated SDC2 in stool DNA samples with concentrations as low as 90 copies/µL in 100% of replicates. The sensitivity for detecting CRC by methylated SDC2-A alone was 85.29% (95% CI 77.03-91.00%) with a specificity of 96.15% (95% CI 91.08-98.58%). The sensitivity by methylated SDC2-B alone was 83.33% (95% CI 74.82-89.42%) with a specificity of 97.69% (95% CI 93.14-99.51%). However, when methylated SDC2-A and methylated SDC2-B were combined, the sensitivity for CRC detection improved to 87.25% (95% CI 79.27-92.53%) with a specificity of 94.62% (95% CI 89.11-97.56%). Further, the detection reagent achieved ROC-AUC 0.874 (95% CI 0.822-0.927) for SDC2-A, 0.906 (95% CI 0.859-0.952) for SDC2-B, and 0.939 (95% CI 0.902-0.977) for SDC2-Combine A&B. CONCLUSIONS: This study validated the capability of stool DNA-based SDC2 methylation test for early screening of CRC, and combined detection of two fragments of SDC2 gene could improve detection sensitivity.

Downregulation of lncRNA SNHG16 inhibits vascular smooth muscle cell proliferation and migration in cerebral atherosclerosis by targeting the miR-30c-5p/SDC2 axis.

Atherosclerosis (AS) is the basic lesion underlying the occurrence and development of cerebrovascular diseases. Abnormal proliferation of vascular smooth muscle cells (VSMCs) plays a crucial role in AS. We aimed to explore the role of SNHG16 in AS and the molecular mechanism of VSMC involvement in the regulation of AS. The expression levels of SNHG16, miR-30c-5p and SDC2 were detected by qRT-PCR. CCK-8, wound healing and Transwell assays were used to assess ox-LDL-induced VSMC proliferation, migration, and invasion, respectively. Western blot analysis was used to detect SDC2 and MEK/ERK pathway-related protein levels. A dual-luciferase reporter assay confirmed the binding of SNHG16 with miR-30c-5p and miR-30c-5p with SDC2. SNHG16 and SDC2 expression was upregulated in patients with AS and ox-LDL-induced VSMCs, while miR-30c-5p was downregulated. Ox-LDL-induced VSMC proliferation and migration were increased, and the MEK/ERK signalling pathway was activated. MiR-30c-5p was targeted to SNHG16 and SDC2. Downregulating SNHG16 or upregulating miR-30c-5p inhibited ox-LDL-induced VSMC proliferation and migration and inhibited MEK/ERK signalling pathway activation. In contrast, downregulating miR-30c-5p or upregulating SDC2 reversed the effects of downregulating SNHG16 or upregulating miR-30c-5p. Furthermore, downregulating SDC2 inhibited ox-LDL-induced proliferation and migration of VSMCs and inhibited activation of the MEK/ERK signalling pathway, while upregulating lncRNA SNHG16 reversed the effects of downregulating SDC2. Downregulation of SNHG16 inhibited VSMC proliferation and migration in AS by targeting the miR-30c-5p/SDC2 axis. This study provides a possible therapeutic approach to AS.

The methylation of SDC2 and TFPI2 defined three methylator phenotypes of colorectal cancer.

BACKGROUND: Methylated SDC2 and TFPI2 are widely used for colorectal cancer (CRC) detection. However, they often miss some CRCs, which directly diminishes the sensitivity. Further investigations of the underlying mechanisms leading to the missed samples will facilitate developing more eligible methylation markers. METHODS: CRC samples from TCGA and GEO datasets were divided into three groups, High-methylation/ High-methylation (HH), High-methylation/Low-methylation (HL), and Low-methylation/Low-methylation (LL) according to the methylation status of SDC2 and TFPI2 promoters. Variations in age, tumor location and microsatellite instable were then assessed between the three groups and verified in our custom cohort. RESULTS: Samples of HL group preferred to derive from left-sided CRCs (P < 0.05). HH samples showed the highest microsatellite instability and mutation load (mean nonsynonymous mutations for HH/HL/LL: 10.55/3.91/7.02, P = 0.0055). Almost all mutations of BRAF, one of the five typical CpG island methylator phenotype (CIMP) related genes, were observed in HH group (HH/HL/LL: 51/0/1, P = 0.018). Besides, older patients were frequently found in HH group. Expression analysis identified 37, 84, and 22 group-specific differentially expressed genes (DEGs) for HH, HL, and LL, respectively. Functional enrichment analysis revealed that HH-specific DEGs were mainly related to transcription regulation, while LL-specific DEGs were enriched in the biological processes of extracellular matrix interaction and cell migration. CONCLUSIONS: The current study revealed that the performance of methylation-based markers might be affected by tumor location, patient age, mutation load and MSI, and these respective sides should be considered when developing new methylation markers for CRC detection.

Shed syndecan-2 enhances colon cancer progression by increasing cooperative angiogenesis in the tumor microenvironment.

Although shed syndecan-2 potentiated the tumorigenic activities of colon cancer cells, how shed syndecan-2 increases this tumorigenic potential remains unclear. Using an orthotopic mouse model of colon cancer, we show that shed syndecan-2 increases colon cancer progression by cooperatively promoting angiogenesis. Co-administration with a synthetic peptide of shed syndecan-2 (S2LQ) enhanced the survival and tumor engraftment of luciferase-expressing CT26 colon adenocarcinoma cells orthotopically implanted into the cecum of BALB/c mice. Intravenous injection of S2LQ further enhanced the growth of orthotopic tumors in the cecum, with increases in the tissue infiltration of macrophages and the formation of blood vessels, mainly in peripheral layers of the tumor facing the stroma. Furthermore, S2LQ stabilized HIF1α and enhanced the VEGF expression in human colon cancer cell lines, and increased the migration of RAW 264.7 murine macrophage cells and bone marrow-derived macrophages. Finally, S2LQ increased the tube formation of vascular endothelial cells in vitro. Together, these data demonstrate that shed syndecan-2 enhances tumorigenic activity by increasing the crosstalk of cancer cells with tumor-associated macrophages and endothelial cells to enhance angiogenesis for colon cancer progression in the tumor microenvironment.

Syndecan-2 regulates PAD2 to exert antifibrotic effects on RA-ILD fibroblasts.

Rheumatoid arthritis (RA)-associated interstitial lung disease (RA-ILD) is the most common pulmonary complication of RA, increasing morbidity and mortality. Anti-citrullinated protein antibodies have been associated with the development and progression of both RA and fibrotic lung disease; however, the role of protein citrullination in RA-ILD remains unclear. Here, we demonstrate that the expression of peptidylarginine deiminase 2 (PAD2), an enzyme that catalyzes protein citrullination, is increased in lung homogenates from subjects with RA-ILD and their lung fibroblasts. Chemical inhibition or genetic knockdown of PAD2 in RA-ILD fibroblasts attenuated their activation, marked by decreased myofibroblast differentiation, gel contraction, and extracellular matrix gene expression. Treatment of RA-ILD fibroblasts with the proteoglycan syndecan-2 (SDC2) yielded similar antifibrotic effects through regulation of PAD2 expression, phosphoinositide 3-kinase/Akt signaling, and Sp1 activation in a CD148-dependent manner. Furthermore, SDC2-transgenic mice exposed to bleomycin-induced lung injury in an inflammatory arthritis model expressed lower levels of PAD2 and were protected from the development of pulmonary fibrosis. Together, our results support a SDC2-sensitive profibrotic role for PAD2 in RA-ILD fibroblasts and identify PAD2 as a promising therapeutic target of RA-ILD.

Syndecan-2 enriches for hematopoietic stem cells and regulates stem cell repopulating capacity.

The discovery of novel hematopoietic stem cell (HSC) surface markers can enhance understanding of HSC identity and function. We have discovered a population of primitive bone marrow (BM) HSCs distinguished by their expression of the heparan sulfate proteoglycan Syndecan-2, which serves as both a marker and a regulator of HSC function. Syndecan-2 expression was increased 10-fold in CD150+CD48-CD34-c-Kit+Sca-1+Lineage- cells (long-term HSCs [LT-HSCs]) compared with differentiated hematopoietic cells. Isolation of BM cells based solely on syndecan-2 surface expression produced a 24-fold enrichment for LT-HSCs and sixfold enrichment for α-catulin+c-kit+ HSCs, and yielded HSCs with superior in vivo repopulating capacity compared with CD150+ cells. Competitive repopulation assays revealed the HSC frequency to be 17-fold higher in syndecan-2+CD34-KSL cells compared with syndecan-2-CD34-KSL cells and indistinguishable from CD150+CD34-KSL cells. Syndecan-2 expression also identified nearly all repopulating HSCs within the CD150+CD34-KSL population. Mechanistically, syndecan-2 regulates HSC repopulating capacity through control of expression of Cdkn1c (p57) and HSC quiescence. Loss of syndecan-2 expression caused increased HSC cell cycle entry, downregulation of Cdkn1c, and loss of HSC long-term repopulating capacity. Syndecan-2 is a novel marker of HSCs that regulates HSC repopulating capacity via control of HSC quiescence.

Mesenchymal stromal cell-derived syndecan-2 regulates the immune response during sepsis to foster bacterial clearance and resolution of inflammation.

Sepsis is a life-threatening process related to a dysregulated host response to an underlying infection, which results in organ dysfunction and poor outcomes. Therapeutic strategies using mesenchymal stromal cells (MSCs) are under investigation for sepsis, with efforts to improve cellular utility. Syndecan (SDC) proteins are transmembrane proteoglycans involved with cellular signaling events including tissue repair and modulating inflammation. Bone marrow-derived human MSCs express syndecan-2 (SDC2) at a level higher than other SDC family members; thus, we explored SDC2 in MSC function. Administration of human MSCs silenced for SDC2 in experimental sepsis resulted in decreased bacterial clearance, and increased tissue injury and mortality compared with wild-type MSCs. These findings were associated with a loss of resolution of inflammation in the peritoneal cavity, and higher levels of proinflammatory mediators in organs. MSCs silenced for SDC2 had a decreased ability to promote phagocytosis of apoptotic neutrophils by macrophages in the peritoneum, and also a diminished capability to convert macrophages from a proinflammatory to a proresolution phenotype via cellular or paracrine actions. Extracellular vesicles are a paracrine effector of MSCs that may contribute to resolution of inflammation, and their production was dramatically reduced in SDC2-silenced human MSCs. Collectively, these data demonstrate the importance of SDC2 for cellular and paracrine function of human MSCs during sepsis.