Enhanced Detection of Genitourinary Cancers Using Fragmentation and Copy Number Profiles Obtained from Urinary Cell-Free DNA.

BACKGROUND: Recent studies have reported that examining the fragmentation profiles (FP) of plasma cell-free DNA (cfDNA) further improves the clinical sensitivity of tumor detection. We hypothesized that considering the differences of the FP of urinary cfDNA would increase the clinical sensitivity of genitourinary (GU) cancer detection. METHODS: 177 patients with GU cancer and 94 individuals without tumors were enrolled in the discovery cohort. An independent validation dataset comprising 30 patients without tumors and 66 patients with GU cancer was also collected. We constructed an ensemble classifier, GUIDER, to detect and localize GU cancers using fragmentation and copy number profiles obtained from shallow whole-genome sequencing of urinary cfDNA. RESULTS: Urinary cfDNA of patients with GU cancer had a higher proportion of long fragments (209-280 bp) and a lower proportion of short fragments (140-208 bp) compared to controls. The overall mean classification accuracy of the FP was 74.62%-85.39% for different algorithms, and integration of the FP and copy number alteration (CNA) features further enhanced the classification of samples from patients with GU cancer. The mean diagnostic accuracy was further improved by the ensemble classifier GUIDER, which integrated the FP and CNA profiles and resulted in a higher mean accuracy (87.52%) compared to the analysis performed without FP features (74.62%). GUIDER performed well in an independent validation dataset. CONCLUSIONS: The lengthening and shortening of urinary cfDNA within specific size ranges were identified in patients with GU cancer. Integration of the FP should further enhance the ability to use urinary cfDNA as a molecular diagnostic tool.

Urothelial Carcinoma Detection Based on Copy Number Profiles of Urinary Cell-Free DNA by Shallow Whole-Genome Sequencing.

BACKGROUND: Current noninvasive assays for urothelial carcinoma (UC) lack clinical sensitivity and specificity. Given the utility of plasma cell-free DNA (cfDNA) biomarkers, the development of urinary cfDNA biomarkers may improve the diagnostic sensitivity. METHODS: We assessed copy number alterations (CNAs) by shallow genome-wide sequencing of urinary cfDNA in 95 cancer-free individuals and 65 patients with UC, 58 with kidney cancer, and 45 with prostate cancer. We used a support vector machine to develop a diagnostic classifier based on CNA profiles to detect UC (UCdetector). The model was further validated in an independent cohort (52 patients). Genome sequencing data of tumor specimens from 90 upper tract urothelial cancers (UTUCs) and CNA data for 410 urothelial carcinomas of bladder (UCBs) from The Cancer Genome Atlas were used to validate the classifier. Genome sequencing data for urine sediment from 32 patients with UC were compared with cfDNA. To monitor the treatment efficacy, we collected cfDNA from 7 posttreatment patients. RESULTS: Urinary cfDNA was a more sensitive alternative to urinary sediment. The UCdetector could detect UC at a median clinical sensitivity of 86.5% and specificity of 94.7%. UCdetector performed well in an independent validation data set. Notably, the CNA features selected by UCdetector were specific markers for both UTUC and UCB. Moreover, CNA changes in cfDNA were consistent with the treatment effects. Meanwhile, the same strategy could localize genitourinary cancers to tissue of origin in 70.1% of patients. CONCLUSIONS: Our findings underscore the potential utility of urinary cfDNA CNA profiles as a basis for noninvasive UC detection and surveillance.

Restoration of 5-hydroxymethylcytosine by ascorbate blocks kidney tumour growth.

Loss of 5-hydroxymethylcytosine (5hmC) occurs frequently in a wide variety of tumours, including clear-cell renal cell carcinoma (ccRCC). It remains unknown, however, whether the restoration of 5hmC patterns in tumours could have therapeutic efficacy. Here, we used sodium L-ascorbate (vitamin C, AsANa) and the oxidation-resistant form L-ascorbic acid 2-phosphate sesquimagnesium (APM) for the restoration of 5hmC patterns in ccRCC cells. At physiological concentrations, both show anti-tumour efficacy during long-term treatment in vitro and in vivo Strikingly, global 5hmC patterns in ccRCC cells after treatment resemble those of normal kidney tissue, which is observed also in treated xenograft tumours, and in primary cells from a ccRCC patient. Further, RNA-seq data show that long-term treatment with vitamin C changes the transcriptome of ccRCC cells. Finally, APM treatment induces less non-specific cell damage and shows increased stability in mouse plasma compared to AsANa. Taken together, our study provides proof of concept for an epigenetic differentiation therapy of ccRCC with vitamin C, especially APM, at low doses by 5hmC reprogramming.

Generation and characterization of a breast carcinoma model by PyMT overexpression in mammary epithelial cells of tree shrew, an animal close to primates in evolution.

The tree shrew is becoming an attractive experimental animal model for human breast cancer owing to a closer relationship to primates/humans than rodents. Tree shrews are superior to classical primates because tree shrew are easier to manipulate, maintain and propagate. It is required to establish a high-efficiency tree shrew breast cancer model for etiological research and drug assessment. Our previous studies suggest that 7,12-dimethylbenz(a)anthracene (DMBA) and medroxyprogesterone acetate (MPA) induce breast tumors in tree shrews with a low frequency (<50%) and long latency (∼ 7-month), making these methods less than ideal. We induced mammary tumors in tree shrew (Tupaia belangeri chinensis) by injection of lentivirus expressing the PyMT oncogene into mammary ducts of 22 animals. Most tree shrews developed mammary tumors with a latency of about three weeks, and by 7 weeks all injected tree shrews had developed mammary tumors. Among these, papillary carcinoma is the predominant tumor type. One case showed lymph node and lung metastasis. Interestingly, the expression levels of phosphorylated AKT, ERK and STAT3 were elevated in 41-68% of PyMT-induced mammary tumors, but not all tumors. Finally, we observed that the growth of PyMT-induced tree shrew mammary tumors was significantly inhibited by Cisplatin and Epidoxorubicin. PyMT-induced tree shrew mammary tumor model may be suitable for further breast cancer research and drug development, due to its high efficiency and short latency.

Tobacco carcinogen NNK-induced lung cancer animal models and associated carcinogenic mechanisms.

Tobacco usage is a major risk factor in the development, progression, and outcomes for lung cancer. Of the carcinogens associated with lung cancer, tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is among the most potent ones. The oncogenic mechanisms of NNK are not entirely understood, hindering the development of effective strategies for preventing and treating smoking-associated lung cancers. Here, we introduce the NNK-induced lung cancer animal models in different species and its potential mechanisms. Finally, we summarize several chemopreventive agents developed from these animal models.

Acute depletion of BRG1 reveals its primary function as an activator of transcription.

The mammalian SWI/SNF-like BAF complexes play critical roles during animal development and pathological conditions. Previous gene deletion studies and characterization of human gene mutations implicate that the complexes both repress and activate a large number of genes. However, the direct function of the complexes in cells remains largely unclear due to the relatively long-term nature of gene deletion or natural mutation. Here we generate a mouse line by knocking in the auxin-inducible degron tag (AID) to the Smarca4 gene, which encodes BRG1, the essential ATPase subunit of the BAF complexes. We show that the tagged BRG1 can be efficiently depleted by osTIR1 expression and auxin treatment for 6 to 10 h in CD4 + T cells, hepatocytes, and fibroblasts isolated from the knock-in mice. The acute depletion of BRG1 leads to decreases in nascent RNAs and RNA polymerase II binding at a large number of genes, which are positively correlated with the loss of BRG1. Further, these changes are correlated with diminished accessibility at DNase I Hypersensitive Sites (DHSs) and p300 binding. The acute BRG1 depletion results in three major patterns of nucleosome shifts leading to narrower nucleosome spacing surrounding transcription factor motifs and at enhancers and transcription start sites (TSSs), which are correlated with loss of BRG1, decreased chromatin accessibility and decreased nascent RNAs. Acute depletion of BRG1 severely compromises the Trichostatin A (TSA) -induced histone acetylation, suggesting a substantial interplay between the chromatin remodeling activity of BRG1 and histone acetylation. Our data suggest BRG1 mainly plays a direct positive role in chromatin accessibility, RNAPII binding, and nascent RNA production by regulating nucleosome positioning and facilitating transcription factor binding to their target sites.

Ascorbic acid induced TET2 enzyme activation enhances cancer immunotherapy efficacy in renal cell carcinoma.

Exploring the regulatory mechanism of PD-L1 in renal cancer is one of the key strategies to improve the response of renal cancer patients to checkpoint blockade therapy. In this study, the synergistic effect of ascorbic acid (vitamin C) supplementation and the impact of TET2 depletion on anti-PD-L1 therapy were determined in xenograft model experiments. Lymphocyte infiltration and chemokine expression were determined using flow cytometry and qRT-PCR. To determine the downstream targets of TET2, we performed hMeDip-seq and RNA-seq analyses. The molecular mechanism was further confirmed by hMeDip-qPCR, MeDip-qPCR, bisulfite sequencing, Western blotting, qRT-PCR and xenograft model experiments in vitro and in vivo. The present study demonstrated that ascorbic acid enhanced the efficacy of immunotherapy and that the loss of TET2 function enabled renal cancer cells to evade antitumor immunity. Ascorbic acid treatment significantly increased the intratumoral infiltration of T cells and the expression of cytokines and chemokines, while the loss of TET2 impaired the infiltration of T cells and the expression of cytokines and chemokines. TET2 was recruited to IRF1 by IFN-γ-STAT1 signaling, thereby maintaining IRF1 demethylation and ultimately inducing PD-L1 expression. These results suggest a new strategy of stimulating TET activity to improve immunotherapy for renal cell carcinoma.

Single-Cell RNA-seq Reveals a Developmental Hierarchy Super-Imposed Over Subclonal Evolution in the Cellular Ecosystem of Prostate Cancer.

Prostate cancer (PCa) is a complex disease. An ongoing accumulation of mutations results in increased genetic diversity, with the tumor acquiring distinct subclones. However, non-genetic intra-tumoral heterogeneity, the cellular differentiation state and the interplay between subclonal evolution and transcriptional heterogeneity are poorly understood. Here, the authors perform single-cell RNA sequencing from 14 untreated PCa patients. They create an extensive cell atlas of the PCa patients and mapped developmental states onto tumor subclonal evolution. They identify distinct subclones across PCa patients and then stratify tumor cells into four transcriptional subtypes, EMT-like (subtype 0), luminal A-like (subtype 1), luminal B/C-like (subtype 2), and basal-like (subtype 3). These subtypes are hierarchically organized into stem cell-like and differentiated status. Strikingly, multiple subclones within a single primary tumor present with distinct combinations of preferential subtypes. In addition, subclones show different communication strengths with other cell types within the tumor ecosystem, which may modulate the distinct transcriptional subtypes of the subclones. Notably, by integrating TCGA data, they discover that both tumor cell transcriptional heterogeneity and cellular ecosystem diversity correlate with features of a poor prognosis. Collectively, their study provides the analysis of subclonal and transcriptional heterogeneity and its implication for patient prognosis.

Construction of diagnostic and subtyping models for renal cell carcinoma by genome-wide DNA methylation profiles.

BACKGROUND: Renal cell carcinoma (RCC) is one of the most common urological cancers and has a poor prognosis. RCC is classified into several subtypes, among which kidney renal clear cell carcinoma (KIRC) and kidney renal papillary cell carcinoma (KIRP) are the two most common subtypes. Due to the lack of adequate screening and comparative analysis of RCC subtypes, effective diagnosis and treatment strategies have not yet been achieved. METHODS: In this study, 450K methylation array data were collected from The Cancer Genome Atlas (TCGA). The 'limma moderated t-test' and LASSO were used to construct diagnostic and subtyping models, and survival analysis was conducted online by GEPIA. RESULTS: We built a model with 15 methylation sites, which showed high diagnostic and subtyping performance in specificity and sensitivity. At the same time, for potential clinical usability, we calculated the diagnostic and subtyping scores to classify RCC from normal tissue and distinguish the different RCC subtypes. Additionally, the CpG sites were mapped to their corresponding genes, which could also be used to predict the prognosis of RCC. CONCLUSIONS: Different methylation sites can be used as diagnostic and subtyping markers that are specific to RCC and RCC subtypes (KIRC and KIRP) with high sensitivity and accuracy.

Identification of plasma secreted phosphoprotein 1 as a novel biomarker for upper tract urothelial carcinomas.

The key prognostic factor at the time of diagnosis of upper tract urothelial carcinomas (UTUC) is whether the tumor is in the muscle-invasive or non-muscle invasive stage. It is critical to identify novel molecular biomarkers for early detection and target therapy. Plasma proteins secreted by tumor tissues have excellent potential as biomarkers for UTUC. In this study, we conducted a systematic study to identify plasma markers for UTUC based on RNA-seq data from five UTUC tissues and paired adjacent noncancerous mucosa. Through bioinformatics analysis, we found secreted phosphoprotein 1 (SPP1) was the most significant gene that coding secretory protein. Then, qRT-PCR and enzyme-linked immunosorbent assay were performed to evaluate the expression and clinical significance of SPP1 in UTUC. Results found that SPP1 mRNA was upregulated in UTUC cells and tissues, and high SPP1 mRNA expression level was closely related to advanced stage and high grade. Moreover, it is suggested that plasma SPP1 may be a potential biomarker to help identify early-stage UTUC patients and predict invasive and high-grade UTUC. In conclusion, plasma SPP1 is a novel biomarker for UTUC.

Diagnostic and prognostic biomarkers of common urological cancers based on aberrant DNA methylation.

AIM: We intended to construct DNA methylation-based models for the diagnosis and prognosis of three common urological cancers including prostate adenocarcinoma, renal clear cell carcinoma and bladder urothelial carcinoma. MATERIALS & METHODS: Total 450K methylation array data from the cancer genome atlas and gene expression omnibus datasets were downloaded. Moderated t-statistics and least absolute shrinkage and selection operator method were used to build diagnosis and prognosis models. RESULTS: Our diagnostic panels including 128 CpG sites had high sensitivity and accuracy in distinguishing samples and could identify lymphatic metastases in prostate adenocarcinoma patients. The prognostic models with 19 CpG sites for renal clear cell carcinoma and 21 CpG sites for bladder urothelial carcinoma were able to distinguish high- and low-risk patients and improve the predictive ability of the tumor node metastasis staging system. CONCLUSION: DNA methylation may afford reliable biomarkers in the diagnosis and prognosis of common urological cancers.

Vitamin C increases 5-hydroxymethylcytosine level and inhibits the growth of bladder cancer.

BACKGROUND: 5-Hydroxymethylcytosine (5hmC) is converted from 5-methylcytosine (5mC) by a group of enzymes termed ten-eleven translocation (TET) family dioxygenases. The loss of 5hmC has been identified as a hallmark of most types of cancer and is related to tumorigenesis and progression. However, the role of 5hmC in bladder cancer is seldom investigated. Vitamin C was recently reported to induce the generation of 5hmC by acting as a cofactor for TET dioxygenases. In this study, we explored the role of 5hmC in bladder cancer and the therapeutic efficacy of vitamin C in increasing the 5hmC pattern. RESULTS: 5hmC was decreased in bladder cancer samples and was related to patient overall survival. Genome-wide mapping of 5hmC in tumor tissues and vitamin C-treated bladder cancer cells revealed that 5hmC loss was enriched in cancer-related genes and that vitamin C treatment increased 5hmC levels correspondingly. Vitamin C treatment shifted the transcriptome and inhibited the malignant phenotypes associated with bladder cancer cells in both in vitro cell lines and in vivo xenografts. CONCLUSIONS: This study provided mechanistic insights regarding the 5hmC loss in bladder cancer and a rationale for exploring the therapeutic use of vitamin C as a potential epigenetic treatment for bladder cancer.

Characterization and phylogenetic analysis of Krüppel-like transcription factor (KLF) gene family in tree shrews (Tupaia belangeri chinensis).

Krüppel-like factors (KLFs) are a family of zinc finger transcription factors regulating embryonic development and diseases. The phylogenetics of KLFs has not been studied in tree shrews, an animal lineage with a closer relationship to primates than rodents. Here, we identified 17 KLFs from Chinese tree shrew (Tupaia belangeri chinensis). KLF proteins are highly conserved among humans, monkeys, rats, mice and tree shrews compared to zebrafish and chickens. The CtBP binding site, Sin3A binding site and nuclear localization signals are largely conserved between tree shrews and human beings. Tupaia belangeri (Tb) KLF5 contains several conserved post-transcriptional modification motifs. Moreover, the mRNA and protein expression patterns of multiple tbKLFs are tissue-specific . TbKLF5, like hKLF5, significantly promotes NIH3T3 cell proliferation in vitro. These results provide insight for future studies regarding the structure and function of the tbKLF gene family.

BAP1 promotes breast cancer cell proliferation and metastasis by deubiquitinating KLF5.

The transcription factor KLF5 is highly expressed in basal-like breast cancer and promotes breast cancer cell proliferation, survival, migration and tumour growth. Here we show that, in breast cancer cells, KLF5 is stabilized by the deubiquitinase (DUB) BAP1. With a genome-wide siRNA library screen of DUBs, we identify BAP1 as a bona fide KLF5 DUB. BAP1 interacts directly with KLF5 and stabilizes KLF5 via deubiquitination. KLF5 is in the BAP1/HCF-1 complex, and this newly identified complex promotes cell cycle progression partially by inhibiting p27 gene expression. Furthermore, BAP1 knockdown inhibits tumorigenicity and lung metastasis, which can be rescued partially by ectopic expression of KLF5. Collectively, our findings not only identify BAP1 as the DUB for KLF5, but also reveal a critical mechanism that regulates KLF5 expression in breast cancer. Our findings indicate that BAP1 could be a potential therapeutic target for breast and other cancers.

PTEN/PIK3CA genes are frequently mutated in spontaneous and medroxyprogesterone acetate-accelerated 7,12-dimethylbenz(a)anthracene-induced mammary tumours of tree shrews.

Tree shrew has increasingly become an attractive experimental animal model for human diseases, particularly for breast cancer due to spontaneous breast tumours and their close relationship to primates and by extension to humans. However, neither normal mammary glands nor breast tumours have been well characterised in the Chinese tree shrew (Tupaia belangeri chinensis). In this study, normal mammary glands from four different developmental stages and 18 spontaneous breast tumours were analysed. Haematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) showed that normal mammary gland morphology and structures of tree shrews were quite similar to those found in humans. Spontaneous breast tumours of tree shrews were identified as being intraductal papilloma, papillary carcinoma, and invasive ductal carcinoma with or without lung metastasis. To further analyse breast cancer tumours among tree shrews, 40 3-4 month-old female tree shrews were orally administrated 20 mg 7,12-dimethylbenz(a)anthracene (DMBA) or peanut oil thrice, and then, 15 of these DMBA administrated tree shrews were implanted with medroxyprogesterone acetate (MPA) pellets. DMBA was shown to induce breast tumours (12%) while the addition of MPA increased the tumour incidence (50%). Of these, three induced breast tumours were intraductal papillary carcinomas and one was invasive ductal carcinoma (IDC). The PTEN/PIK3CA (phosphatase and tensin homologue/phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha), but not TP53 and GATA3, genes are frequently mutated in breast tumours, and the PTEN/PIK3CA gene mutation status correlated with the expression of pAKT in tree shrew breast tumours. These results suggest that tree shrews may be a promising animal model for a subset of human breast cancers with PTEN/PIK3CA gene mutations.