Abstract Background: Methylation of the MGMT gene promoter was observed in approximately 50% of glioblastoma multiforme (GBM). Epigenetic silencing of the MGMT gene by promoter methylation results in decreased MGMT protein expression, reduced DNA repair activity, and potential increased sensitivity to alkylating agent-based chemotherapy. Although MGMT promoter methylation status has been widely evaluated by methylation-specific PCR and bisulfite pyrosequencing. The limitations to these methods include low quantitative accuracy and low sample throughput. Here, we developed a high throughput method (in-house One-Step Seq Method, Genetron) which combines bisulfite conversion with amplicon sequencing of MGMT gene promoter. Methods: DNA was extracted from 144 FFPE or fresh frozen glioma tissues. For bisulfite pyrosequencing, MGMT promoter methylation status was analyzed using the MGMT Pyro Kit (Qiagen 970061) following the manufacturer's protocol. At the same time,the MGMT promoter methylation status was also analyzed by in-house One-Step Seq Methodfollowing bisulfite conversion. In briefly, the DNA was pre-treated by sodium bisulfite. Then Exon 1 of human MGMT gene was amplified from bisulfite conversed DNA. And library was constructed at the same time. High-throughput sequencing was performed on Ion GeneStudio S5. Reads mapping and methylation calling was handled by Bismark. The DNA sample was identified as MGMT methylation, if average methylation level of all CpG sites in exon 1 of MGMT was more than 21 %. Results: Currently, bisulfite pyrosequencing was considered as the gold standard for DNA methylation analysis. Comparing the result of bisulfite pyrosequencing analysis with the result of in-house methylation status analysis, 94.4% of DNA samples from 144 glioma tissues showed consistent methylation status of MGMT gene promotor. 5 DNA samples were identified as non-methylation in bisulfite pyrosequencing, but with methylation in in-house methylation status analysis. While 3 DNA samples were identified as methylation in bisulfite pyrosequencing, but not in in-house methylation status analysis. Meanwhile, by in-house One-Step Seq Method following bisulfite conversion, both the bisulfite conversion and library construction could be completed injust 4 hours using 10 ng DNA, and MGMT gene promoter methylation status could be estimated in two days. Conclusions: We developed a High-throughput sequencing based method to analyze MGMT status accurately combines bisulfite conversion with amplicon sequencing. This method shows high accuracy, high throughput, and easy manipulation for molecular classification of brain cancer. Citation Format: Yukun Zhang, Min Shi, Qiaosong Zheng, Xiao Shi, Min Chen, Le Li, Huiming Zhu, Liping Jiang, Tonghui Ma, Sumin Geng. Methylation MGMT gene promoter analysis based on a high throughput method combines bisulfite conversion with amplicon sequencing [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3186.
Wt1 encodes a zinc finger nuclear transcriptional factor, which is specifically expressed in testicular Sertoli cells and knockdown of Wt1 in Sertoli cells causes male mice subfertility. However, the underlying mechanism is still unclear. In this study, we found that expression of inhibin-α is significantly reduced in Wt1-deficient Sertoli cells. Luciferase assays using the inhibin-α promoter indicated that the inhibin-α promoter is transactivated by the Wt1 A, and B isoforms (-KTS), but not the C, and D isoforms (+KTS). Analysis of the Wt1 responsive element of the inhibin-α promoter region using site-directed mutagenesis showed that the nucleotides between -58 and -49 are essential for Wt1-dependent transactivation of the inhibin-α promoter. ChIP assays indicated that Wt1 directly interacts with the inhibin-α promoter. In addition, the inhibin-α promoter is activated synergistically by Wt1 and Sf1. Mutation of the ligand binding domain (LBD) of Sf1 (residues 235-238) completely abolished the synergistic action between Wt1 and Sf1, but did not affect the physical interaction between these two proteins, suggesting that other factor(s) may also be involved in the regulation of inhibin-α in Sertoli cells. Further studies demonstrated that β-catenin enhances the synergistic activation of Wt1 and Sf1 on the inhibin-α promoter. Given the fact that inhibin-α, a subunit of inhibin, is known to be involved in the regulation of spermatogenesis and testicular steroidogenesis, this study reveals a new regulatory mechanism of inhibin-α in Sertoli cells and also sheds light on the physiological functions of Wt1 in gonad development and spermatogenesis.
Immune checkpoint inhibitors (ICIs) have become important treatment strategies, yet responses vary among patients and predictive biomarkers are urgently needed. Mutations in KMT2C and KMT2D lead to increased levels of genomic instability. Therefore, we aimed to examine whether KMT2C/D mutations might be a predictor of immunotherapeutic efficacy. Here, we investigated the associations of KMT2C/D loss-of-function (LOF) variants with tumor mutation burden (TMB), MSI-H, PD-L1 expression, the levels of tumor-infiltrating leukocytes (TILs), and clinical response to ICIs. It was found that KMT2C/D LOF variants were associated with higher TMB. Compared with the non-LOF group, the proportion of patients with MSI-H tumors was larger in the LOF group. PD-L1 expression was higher in the LOF group only for colorectal cancer in both the Chinese and The Cancer Genome Atlas cohorts. Importantly, KMT2C/D LOF variants were associated with decreased regulatory T cells and increased levels of CD8+ T cells, activated NK cells, M1 macrophages, and M2 macrophages in colorectal cancer. However, there was no significant association between KMT2C/D LOF and TILs levels in other cancer types. Consistently, the results showed that KMT2C/D LOF variants were associated with prolonged overall survival only in colorectal cancer (p = 0.0485). We also presented that patients with KMT2C/D LOF mutations exhibited a better clinical response to anti-PD-1 therapy in a Chinese colorectal cancer cohort (p = 0.002). Taken together, these results suggested that KMT2C/D LOF variants could be a useful predictor for ICIs efficacy in colorectal cancer. In addition, the predictive value of KMT2C/D LOF variants was consistent with their association with TILs levels.
Background: In the large amount of hematuria patients, a considerable part of them originate in upper tract urinary carcinoma(UTUC). The most important in the evaluation of hematuria is to find accurate and economical method to discriminate between non-malignant and malignant causes. Liquid biopsy for urine samples can be helpful for UTUC diagnosis in patients with hematuria and rule out individuals with low-risk. Methods: In this study, we combined high-throughput sequencing of 17 genes and methylation analysis for ONECUT2 CpG sites as a liquid biopsy panel. We developed a prediction model which contained several significant features to evaluate the risk of UTUC in hematuria patients. Findings: 94 healthy, 70 UTUC- and 70 UTUC+ cases’ samples are qualified for this study, our liquid biopsy panel resulting in a sensitivity of 94% (66/70), and a specificity of 96% (158/164), the positive predication value of 92% and a negative predication value of 98% in this study. With age as additional factor, our model was able to stratify patients into a low or high-risk group with an accuracy score at 94%. Further validation in a large prospective cohort will answer whether this early screening tool can also be used in monitoring postoperative patients who need serials of follow-ups. Interpretation: Urine based liquid biopsy can assist evaluating upper tract urinary carcinoma risk of patients with hematuria and outperformed FISH for accurately ruling out patients who do not have UTUC. Funding Statement: In this research, no funding or other financial supports was received.Declaration of Interests: The authors declared that they have no conflicts of interest to this study. Ethics Approval Statement: Informed written consent was obtained from patients at PLA General Hospital and the study was approved by the Committees on Clinical Research Ethics of the Chinese PLA General Hospital.
Wt1 is specifically expressed in Sertoli cells in the developing testis. A previous study has demonstrated that Wt1 plays a critical role in maintaining the integrity of testicular cords. However, the underlying mechanism is unclear. In this study, we found that the laminin-positive basal lamina lining the testicular cords was fragmented and completely absent in some areas of Wt1(-/flox); Amh-Cre testes, indicating that the testicular cord disruption can be attributed to the breakdown of the basement membrane. To explore the molecular mechanism underlying this effect, we examined the expression of cell adhesion molecules (CAMs) and testicular cord basal lamina components by real-time RT-PCR, Western blotting, and immunostaining. Compared with control testes, the expression of CAMs (such as E-cadherin, N-cadherin, claudin11, occludin, beta-catenin, and ZO-1) was not obviously altered in Wt1(-/flox); Amh-Cre testes. However, the mRNA level of Col4a1 and Col4a2 was significantly decreased in Wt1-deficient testes. Immunostaining assays further confirmed that the collagen IV protein levels were dramatically reduced in Wt1(-/flox); Amh-Cre testes. Moreover, luciferase and point mutation analyses revealed that the Col4a1 and Col4a2 promoters were additively transactivated by WT1 and SOX9. Given this finding and previous results showing that SOX9 expression declines rapidly after Wt1 deletion, we conclude that the loss of Wt1 in Sertoli cells results in the downregulation of the important basal lamina component, which in turn causes the breakdown of the basal lamina and subsequent testicular cord disruption.
Wilms' tumor 1 (Wt1) is a tumor suppressor gene encoding ∼24 zinc finger transcription factors. In the mammalian testis, Wt1 is expressed mostly by Sertoli cells (SCs) involved in testis development, spermatogenesis, and adult Leydig cell (ALC) steroidogenesis. Global knockout (KO) of Wt1 is lethal in mice due to defects in embryogenesis. Herein, we showed that Wt1 is involved in regulating fetal Leydig cell (FLC) degeneration and ALC differentiation during testicular development. Using Wt1(-/flox);Amh-Cre mice that specifically deleted Wt1 in the SC vs. age-matched wild-type (WT) controls, FLC-like-clusters were found in Wt1-deficient testes that remained mitotically active from postnatal day 1 (P1) to P56, and no ALC was detected at these ages. Leydig cells in mutant adult testes displayed morphological features of FLC. Also, FLC-like cells in adult mutant testes had reduced expression in ALC-associated genes Ptgds, Sult1e1, Vcam1, Hsd11b1, Hsd3b6, and Hsd17b3 but high expression of FLC-associated genes Thbs2 and Hsd3b1. Whereas serum LH and testosterone level in mutant mice were not different from controls, intratesticular testosterone level was significantly reduced. Deletion of Wt1 gene also perturbed the expression of steroidogenic enzymes Star, P450c17, Hsd3b6, Hsd3b1, Hsd17b1, and Hsd17b3. FLCs in adult mutant testes failed to convert androstenedione to testosterone due to a lack of Hsd17b3, and this defect was rescued by coculturing with fetal SCs. In summary, FLC-like cells in mutant testes are putative FLCs that remain mitotically active in adult mice, illustrating that Wt1 dictates the fate of FLC and ALC during postnatal testis development.
Abstract Background The directional migration and the following development of primordial germ cells (PGCs) during gonad formation are key steps for germline development. It has been proposed that the interaction between germ cells and genital ridge (GR) somatic cells plays essential roles in this process. However, the in vivo functional requirements of GR somatic cells in germ cell development are largely unknown. Results Wt1 mutation ( Wt1 R394W/R394W ) results in GR agenesis through mitotic arrest of coelomic epitheliums. In this study, we employed the GR-deficient mouse model, Wt1 R394W/R394W , to investigate the roles of GR somatic cells in PGC migration and proliferation. We found that the number of PGCs was dramatically reduced in GR-deficient embryos at embryonic day (E) 11.5 and E12.5 due to decreased proliferation of PGCs, involving low levels of BMP signaling. In contrast, the germ cells in Wt1 R394W/R394W embryos were still mitotically active at E13.5, while all the germ cells in control embryos underwent mitotic arrest at this stage. Strikingly, the directional migration of PGCs was not affected by the absence of GR somatic cells. Most of the PGCs reached the mesenchyme under the coelomic epithelium at E10.5 and no ectopic PGCs were noted in GR-deficient embryos. However, the precise positioning of PGCs was disrupted. Conclusions Our work provides in vivo evidence that the proliferation of germ cells is precisely regulated by GR somatic cells during different stages of gonad development. GR somatic cells are probably dispensable for the directional migration of PGCs, but they are required for precise positioning of PGCs at the final step of migration.
Scrotal hypothermia is essential for normal spermatogenesis, and temporal heat stress causes a reversible disruption of the blood-testis barrier (BTB). Previous studies have shown that AR expression in primary monkey Sertoli cells (SCs) was dramatically reduced after temporary heat treatment. However, the mechanisms underlying the heat-induced reversible disruption of the BTB, including whether it is directly regulated by the AR, remain largely unknown. In this study, we demonstrated that the AR acts upstream to regulate the heat-induced reversible change in the BTB in mice. When the AR was overexpressed in SCs using an adenovirus, the heat stress-induced down-regulation of BTB-associated proteins (Zonula occludens-1 (ZO-1), N-Cadherin, E-Cadherin, α-Catenin, and β-Catenin) was partially rescued. AR knockdown by RNAi or treatment with flutamide (an AR antagonist) in SCs inhibited the recovery of BTB-associated protein expression after 43°C heat treatment for 30 min. The results of an in vivo AR antagonist injection experiment further showed that the recovery of BTB permeability induced by temporal heat stress was regulated by the AR. Furthermore, we observed that the co-localization and interactions of partitioning-defective protein (Par) 6-Par3-aPKC-Cdc42 polarity complex components were disrupted in both AR-knockdown and heat-induced SCs. AR overexpression in SCs prevented the disruption of these protein-protein interactions after heat treatment. AR knockdown or treatment with flutamide in SCs inhibited the restoration of these protein-protein interactions after heat treatment compared with heat treatment alone. Together, these results demonstrate that the AR plays a crucial role in the heat-induced reversible change in BTB via the Par polarity complex.
Abstract Background: Conventional clinical detection methods such as CT, urine cytology, and ureteroscopy display low sensitivity and/or are invasive in the diagnosis of upper tract urinary carcinoma (UTUC), a factor precluding their use. Previous studies on urine biopsy have not shown satisfactory sensitivity and specificity in the application of both gene mutation or gene methylation panels. Therefore, these unfavorable factors call for an urgent need for a sensitive and non-invasive method for the diagnosis of UTUC. Methods: In this study, a total of 161 hematuria patients were enrolled with (n=69) or without (n=92) UTUC. High-throughput sequencing of 17 genes and methylation analysis for ONECUT2 CpG sites were combined as a liquid biopsy test panel. Further, a logistic regression prediction model that contained several significant features was used to evaluate the risk of UTUC in these patients. Results: In total, 86 UTUC- and 64 UTUC+ case samples were enrolled for the analysis. A logistic regression analysis of significant features including age, the mutation status of TERT promoter and ONECUT2 methylation level resulted inan optimal model with a sensitivity of 94.0%, a specificity of 93.1%, the positive predictive value of 92.2% and a negative predictive value of 94.7%. Notably, the area under the curve (AUC) was 0.957 in the training dataset while internal validation produced an AUC of 0.962. It is worth noting that during follow-up, a patient diagnosed with ureteral inflammation at the time of diagnosis exhibiting both positive mutation and methylation test results was diagnosed with ureteral carcinoma 17 months after his enrollment. Conclusion: This work utilized the epigenetic biomarker ONECUT2 for the first time in the detection of UTUC and discovered its superior performance. To improve its sensitivity, we combined the biomarker with a high-throughput sequencing of 17 genes test. It was found that the selected logistic regression model diagnosed with ureteral cancerThetcan evaluate upper tract urinary carcinoma risk of patients with hematuria and outperform other existing panels in providing clinical recommendations for the diagnosis of UTUC. Moreover, its high negative predictive value is conducive to rule to exclude patients without UTUC.
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