Biology and Clinical Implications of the 19q13 Aggressive Prostate Cancer Susceptibility Locus.

Genome-wide association studies (GWAS) have identified rs11672691 at 19q13 associated with aggressive prostate cancer (PCa). Here, we independently confirmed the finding in a cohort of 2,738 PCa patients and discovered the biological mechanism underlying this association. We found an association of the aggressive PCa-associated allele G of rs11672691 with elevated transcript levels of two biologically plausible candidate genes, PCAT19 and CEACAM21, implicated in PCa cell growth and tumor progression. Mechanistically, rs11672691 resides in an enhancer element and alters the binding site of HOXA2, a novel oncogenic transcription factor with prognostic potential in PCa. Remarkably, CRISPR/Cas9-mediated single-nucleotide editing showed the direct effect of rs11672691 on PCAT19 and CEACAM21 expression and PCa cellular aggressive phenotype. Clinical data demonstrated synergistic effects of rs11672691 genotype and PCAT19/CEACAM21 gene expression on PCa prognosis. These results provide a plausible mechanism for rs11672691 associated with aggressive PCa and thus lay the ground work for translating this finding to the clinic.

Dna2 removes toxic ssDNA-RPA filaments generated from meiotic recombination-associated DNA synthesis.

During the repair of DNA double-strand breaks (DSBs), de novo synthesized DNA strands can displace the parental strand to generate single-strand DNAs (ssDNAs). Many programmed DSBs and thus many ssDNAs occur during meiosis. However, it is unclear how these ssDNAs are removed for the complete repair of meiotic DSBs. Here, we show that meiosis-specific depletion of Dna2 (dna2-md) results in an abundant accumulation of RPA and an expansion of RPA from DSBs to broader regions in Saccharomyces cerevisiae. As a result, DSB repair is defective and spores are inviable, although the levels of crossovers/non-crossovers seem to be unaffected. Furthermore, Dna2 induction at pachytene is highly effective in removing accumulated RPA and restoring spore viability. Moreover, the depletion of Pif1, an activator of polymerase δ required for meiotic recombination-associated DNA synthesis, and Pif1 inhibitor Mlh2 decreases and increases RPA accumulation in dna2-md, respectively. In addition, blocking DNA synthesis during meiotic recombination dramatically decreases RPA accumulation in dna2-md. Together, our findings show that meiotic DSB repair requires Dna2 to remove ssDNA-RPA filaments generated from meiotic recombination-associated DNA synthesis. Additionally, we showed that Dna2 also regulates DSB-independent RPA distribution.

A Digital PCR Method Based on Highly Specific Taq for Detecting Gene Editing and Mutations.

Digital PCR (dPCR) has great potential for assessing gene editing or gene mutation due to its ability to independently inspect each DNA template in parallel. However, current dPCR methods use a fluorescence-labeled probe to detect gene variation events, and their ability to distinguish variated sequences from the wild-type sequence is limited by the probe's tolerance to mismatch. To address this, we have developed a novel dPCR method that uses a primer instead of a probe to sense gene variation. The enhanced Taq DNA polymerase in the PCR system has a high mismatch sensitivity, which enables our dPCR method to distinguish gene mutations from wild-type sequences. Compared to current dPCR methods, our method shows superior precision in assessing gene editing efficiency and single-base DNA mutation. This presents a promising opportunity to advance gene editing research and rare gene mutation detection.

Functional Screenings Identify Regulatory Variants Associated with Breast Cancer Susceptibility.

Genome-wide association studies (GWAS) have identified more than 2000 single nucleotide polymorphisms (SNPs) associated with breast cancer susceptibility, most of which are located in the non-coding region. However, the causal SNPs functioning as gene regulatory elements still remain largely undisclosed. Here, we applied a Dinucleotide Parallel Reporter sequencing (DiR-seq) assay to evaluate 288 breast cancer risk SNPs in nine different breast cancer cell lines. Further multi-omics analysis with the ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing), DNase-seq (DNase I hypersensitive sites sequencing) and histone modification ChIP-seq (Chromatin Immunoprecipitation sequencing) nominated seven functional SNPs in breast cancer cells. Functional investigations show that rs4808611 affects breast cancer progression by altering the gene expression of NR2F6. For the other site, rs2236007, the alteration promotes the binding of the suppressive transcription factor EGR1 and results in the downregulation of PAX9 expression. The downregulated expression of PAX9 causes cancer malignancies and is associated with the poor prognosis of breast cancer patients. Our findings contribute to defining the functional risk SNPs and the related genes for breast cancer risk prediction.

Parallel Reporter Assays Identify Altered Regulatory Role of rs684232 in Leading to Prostate Cancer Predisposition.

Functional characterization of cancer risk-associated single nucleotide polymorphism (SNP) identified by genome-wide association studies (GWAS) has become a big challenge. To identify the regulatory risk SNPs that can lead to transcriptional misregulation, we performed parallel reporter gene assays with both alleles of 213 prostate cancer risk-associated GWAS SNPs in 22Rv1 cells. We disclosed 32 regulatory SNPs that exhibited different regulatory activities with two alleles. For one of the regulatory SNPs, rs684232, we found that the variation altered chromatin binding of transcription factor FOXA1 on the DNA region and led to aberrant gene expression of VPS53, FAM57A, and GEMIN4, which play vital roles in prostate cancer malignancy. Our findings reveal the roles and underlying mechanism of rs684232 in prostate cancer progression and hold great promise in benefiting prostate cancer patients with prognostic prediction and target therapies.

Galangin inhibits tumor growth and metastasis of B16F10 melanoma.

Galangin, an active flavonoid extracted from the root of the Alpinia officinarum Hance, showed a cytotoxic effect on several cancer cell lines in vitro. However, there is no information available concerning its antimetastatic effect. Focal adhesion kinase (FAK), a cytoplasmic tyrosine kinase, is involved in many aspects of cellular processes such as proliferation, adhesion, and invasion. Studies have shown that FAK is a promising target for therapeutic intervention in melanoma. In the present study, proliferation of B16F10 cells was suppressed when exposed to various doses of galangin. Inhibition on proliferation by galangin was also detected by clonogenic survival assay. The capabilities of cell adhesion, cell spreading, and cell motility were impaired by galangin, reinforced by F-actin rearrangement. Molecular data showed that both FAK mRNA level and protein level were reduced dose-dependently. Additionally, galangin reduced phosphorylation of FAK (Tyr397) protein. Transient transfection reporter assays showed that galangin suppressed the transcription of FAK gene, indicating FAK expression is a candidate target of galangin. The antimetastatic function of galangin is further supported by the fact that it could inhibit the formation of tumor colonies in the lung tissue on C57BL/6J mouse lung metastatic model using B16F10 melanoma cells. Immunochemical analyses showed that galangin decreased FAK expression in vivo. These data add to our new understanding that galangin can inhibit B16F10 melanoma metastasis both in vivo and in vitro, and that FAK is a valid therapeutic target against melanoma.

[Synergistic apoptotic effect of the combination of diosgenin and TRAIL on non-small-cell lung cancer cell line A549 evaluated with the Chou-Talalay method].

This study is to investigate the apoptotic induction effect of the combination of diosgenin and TNF-related apoptosis-inducing ligand (TRAIL) on non-small-cell lung cancer cell line A549 by using the Chou-Talalay method, and observe the mechanism of the combination. The apoptotic effect of diosgenin or TRAIL alone and their combination on A549 and normal cell line 293T proliferation was measured by MTT assay. Chou-Talalay method was used to evaluate the combination effect. Apoptosis was examined by Hoechst 33342 staining and flow cytometry assay. Western blotting detects the expression of apoptosis-associated proteins. Diosgenin or TRAIL alone can inhibit proliferation ofA549 in a concentration-dependent manner. According to the Chou-Talalay method, when f(a) = 0.1, CI > 1, when f(a) > 0.1, CI < 1. Combined with TRAIL, the IC50 of diosgenin decreases from 21.864 to 14.810 micromol x L(-1) (P < 0.05) on A549 cells. But for 293T cells, IC50 of diosgenin does not change significantly. As with Hoechst 33342 staining and flow cytometry assay, the apoptosis ratios also increased in the combination group. At protein expression level, combination-treated group displays increased Caspase-8, Caspase-9, Bid, Caspase-3 activation and PARP cleavage, significantly decreased Bcl-2 and increased Bax expression, and MAPK pathways were activated. The combination of diosgenin and TRAIL has synergistic effect on A549 cells.

GPI-anchored glutathione S-transferase as marker allows affinity sorting of transfection-positive cells.

Cell transfection efficiency is still a limiting factor in gene function research. A method that allows isolation and enrichment of the transfection-positive cells is an effective solution. Here, we report a transfection-positive cell sorting system that utilizes GPI-anchored GST (Glutathione S-transferase) as a plasmid marker. The Glutathione S-transferase fusion protein will be expressed and displayed on the cell surface through GPI anchor, and hence permits the positive cells to be isolated using Glutathione (GSH) Magnetic Beads. We prove that the system works efficiently in both the adherent Lenti-X 293T cells and the suspension K-562 cells. The affinity cell sorting procedure efficiently enriched positive cells from 20% to 98% in K-562 cells. The applications in gene knockdown and overexpression experiments in K-562 cells dramatically enhanced the extent of gene alteration, with the gene knockdown efficiency increasing from 7% to 60% and the gene overexpression level rising from 47 to 253 times. This Glutathione S-transferase affinity transfection-positive cell sorting method is simple and fast to operate, large-instrument free, low cost, and hence possesses great potential in gene function study in vitro.

A Gene Transfer-Positive Cell Sorting System Utilizing Membrane-Anchoring Affinity Tag.

Gene delivery efficiency is an essential limit factor in gene study and gene therapy, especially for cells that are hard for gene transfer. Here we develop an affinity cell sorting system that allows efficient enrichment of gene transfer-positive cells. The system expresses an enhanced green fluorescent protein (EGFP) fused with an N-terminal high-affinity Twin-Strep-Tag (TST) that will be anchored to the cell membrane at the out-surface through a glycosylphosphatidylinositol (GPI) membrane-anchoring structure. The EGFP permits microscopy and flow cytometry analysis of the gene transfer-positive cells, and the TST tag at the N terminal of EGFP allows efficient affinity sorting of the positive cells using Strep-Tactin magnetic beads. The cell sorting system enables efficient isolation of gene transfer-positive cells in a simple, convenient, and fast manner. Cell sorting on transfected K-562 cells resulted in a final positive cell percentage of up to 95.0% with a positive cell enrichment fold of 5.8 times. The applications in gene overexpression experiments could dramatically increase the gene overexpression fold from 10 times to 58 times, and in shRNA gene knockdown experiments, cell sorting increased the gene knockdown efficiency from 12% to 53%. In addition, cell sorting in CRISPR/Cas9 genome editing experiments allowed more significant gene modification, with an editing percentage increasing from 20% to 79%. The gene transfer-positive cell sorting system holds great potential for all gene transfer studies, especially on those hard-to-transfect cells.

Increasing Genome Editing Efficiency of Cas9 Nucleases by the Simultaneous Use of Transcriptional Activators and Histone Acetyltransferase Activator.

The CRISPR-Cas9 system shows diverse levels of genome editing activities on eukaryotic chromatin, and high-efficiency sgRNA targets are usually desired in application. In this study, we show that chromatin open status is a pivotal determinant of the Cas9 editing activity in mammalian cells, and increasing chromatin accessibility can efficiently improve Cas9 genome editing. However, the strategy that increases chromatin openness by fusing the VP64 transcriptional activation domain at the C-terminus of Cas9 can only promote genome editing activity slightly at most tested CRISPR-Cas9 targets in Lenti-X 293T cells. Under the enlightenment that histone acetylation increases eukaryotic chromatin accessibility, we developed a composite strategy to further improve genome editing by activating histone acetylation. We demonstrate that promoting histone acetylation using the histone acetyltransferase activator YF-2 can improve the genome editing by Cas9 and, more robustly, by the Cas9 transcriptional activator (Cas9-AD). This strategy holds great potential to enhance CRISPR-Cas9 genome editing and to enable broader CRISPR gRNA target choices for experiments in eukaryotes.

Dinucleotide tag-based parallel reporter gene assay method enables efficient identification of regulatory mutations.

BACKGROUND: The causal single nucleotide polymorphisms (SNPs) leading to increased cancer predisposition mainly function as gene regulatory elements, the evaluation of which largely relies on the parallel reporter gene assay system. However, the common DNA barcodes used in parallel reporter gene assay systems typically because nucleotide composition bias, and many barcodes must be allocated for each sequence to reduce the bias effect. MAIN METHODS AND MAJOR RESULTS: Here, a versatile dinucleotide-tag reporter system (DiR) that enables parallel analysis of regulatory elements with minimized bias based on next-generation sequencing is described. The DiR system is more robust than the classical luciferase assay method, particularly for the investigation of moderate-level regulatory elements. The authors applied the DiR-seq assay in the functional evaluation of SNPs with prostate cancer risk and nominated two and six regulatory SNPs in PC-3 and LNCaP cells, respectively. CONCLUSIONS AND IMPLICATIONS: The DiR system has great potential to advance the functional study of SNPs associated with polygenic disease risks.

Enhanced Taq Variant Enables Efficient Genome Editing Testing and Mutation Detection.

Detection of genome editing with quantitative polymerase chain reaction (PCR) primarily relies on and is limited by its ability to discriminate genome modification from the wild-type sequence. An enhanced DNA polymerase variant with superior specificity is needed for this application. Here, we perform semi-rational molecular evolution on full-length Taq polymerase to screen high-specific variants that meet the requirements of gene variation detection. We substituted each of the 40 polar amino acids in direct contact with the primer/template duplex and conducted extensive random mutagenesis to generate a Taq mutation library. Screening on a quantitative PCR system with insertion and deletion-containing templates identified a series of improved Taq variants. We demonstrate that the Taq388 variant bearing three amino acid substitutions, S577A, W645R, and I707V, has improved sensitivity to insertion and deletion-derived primer/template mismatch by a ΔCt value of 25-26 and is superior for application in evaluating CRISPR-Cas9 editing efficiency and single-cell clone genotyping. In addition, the Taq variant shows substantial potential for single-nucleotide polymorphism detection by means of allele-specific PCR because of its high sensitivity to mismatches.

rs10514231 Leads to Breast Cancer Predisposition by Altering ATP6AP1L Gene Expression.

Numerous genetic variants located in autophagy-related genes have been identified for association with various cancer risks, but the biological mechanisms underlying these associations remain largely unknown. Here we investigated their regulatory activity with a parallel reporter gene assay system in breast cancer cells and identified multiple regulatory SNP sites, including rs10514231. It was located in the second intron of ATG10 and showed gene regulatory activity in most breast cancer cells we used. Mechanistically, the T allele of rs10514231 led to ATP6AP1L downregulation by decreasing the binding affinity of TCF7L2. Overexpression of the ATP6AP1L gene in cancer cells diminished cell proliferation, migration, and invasion. Notably, ATP6AP1L downregulation correlated with breast cancer risk and with poor prognosis in patients. These results provide a plausible mechanism behind the association of rs10514231 with breast cancer risk and will be important for more effective therapeutic target identification for precision medicine.

Large Multicohort Study Reveals a Prostate Cancer Susceptibility Allele at 5p15 Regulating TERT via Androgen Signaling-Orchestrated Chromatin Binding of E2F1 and MYC.

Aberrant telomerase reverse transcriptase (TERT) expression is crucial for tumor survival and cancer cells escaping apoptosis. Multiple TERT-locus variants at 5p15 have been discovered in association with cancer risk, yet the underlying mechanisms and clinical impacts remain unclear. Here, our association studies showed that the TERT promoter variant rs2853669 confers a risk of prostate cancer (PCa) in different ethnic groups. Further functional investigation revealed that the allele-specific binding of MYC and E2F1 at TERT promoter variant rs2853669 associates with elevated level of TERT in PCa. Mechanistically, androgen stimulations promoted the binding of MYC to allele T of rs2853669, thereby activating TERT, whereas hormone deprivations enhanced E2F1 binding at allele C of rs2853669, thus upregulating TERT expression. Notably, E2F1 could cooperate with AR signaling to regulate MYC expression. Clinical data demonstrated synergistic effects of MYC/E2F1/TERT expression or with the TT and CC genotype of rs2853669 on PCa prognosis and severity. Strikingly, single-nucleotide editing assays showed that the CC genotype of rs2853669 obviously promotes epithelial-mesenchymal transition (EMT) and the development of castration-resistant PCa (CRPC), confirmed by unbiased global transcriptome profiling. Our findings thus provided compelling evidence for understanding the roles of noncoding variations coordinated with androgen signaling and oncogenic transcription factors in mis-regulating TERT expression and driving PCa.

A convenient spectrometric assay system for intracellular quantitative measurement of DNA glycosylase activity.

Cytosine methylation is a vital biology event. However, it is also the source of genomic instability due to deamination of 5'-methylcytosine by spontaneous hydrolysis, which produces thymine and results in G:T mismatches. Thymine DNA glycosylase and methyl-CpG-binding protein 4 are major DNA glycosylases involved in the mismatch repair progress, and their activities have been measured in many related researches. In this study, we developed a convenient spectrometric assay system for specific and quantitative measurement of intracellular DNA glycosylase activity. A G:T mismatch was introduced into the upstream region of firefly luciferase-coding sequence in the pGL3-control plasmid. Only if the G:T mismatches were repaired to G:C, will luciferase be expressed in transfected cells. By measuring luciferase activity, which is simple and convenient, the intracellular DNA glycosylase activity can be determined.

Extracellular signal regulated kinase 5 promotes cell migration, invasion and lung metastasis in a FAK-dependent manner.

This study was designed to evaluate ERK5 expression in lung cancer and malignant melanoma progression and to ascertain the involvement of ERK5 signaling in lung cancer and melanoma. We show that ERK5 expression is abundant in human lung cancer samples, and elevated ERK5 expression in lung cancer was linked to the acquisition of increased metastatic and invasive potential. Importantly, we observed a significant correlation between ERK5 activity and FAK expression and its phosphorylation at the Ser910 site. Mechanistically, ERK5 increased the expression of the transcription factor USF1, which could transcriptionally upregulate FAK expression, resulting in FAK signaling activation to promote cell migration. We also provided evidence that the phosphorylation of FAK at Ser910 was due to ERK5 but not ERK1/2, and we then suggested a role for Ser910 in the control of cell motility. In addition, ERK5 had targets in addition to FAK that regulate epithelial-to-mesenchymal transition and cell motility in cancer cells. Taken together, our findings uncover a cancer metastasis-promoting role for ERK5 and provide the rationale for targeting ERK5 as a potential therapeutic approach.

Enhancement of antitumor properties of TRAIL by targeted delivery to the tumor neovasculature.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent with tumor-selective apoptotic activity. TRAIL plays a role in the innate and adaptive immune response and autoimmune disease and may also be involved in hepatic cell death and inflammation. For these reasons, chronic exposure to TRAIL may have deleterious side effects in patients as a cancer therapeutic. In this study, we have improved the antitumor activity of TRAIL by targeted delivery to the tumor vasculature, leading to dramatic enhancement of its therapeutic properties. TRAIL was fused to the ACDCRGDCFC peptide (named RGD-L-TRAIL), a ligand of alpha(V)beta(3) and alpha(V)beta(5) integrins. Biological activity was evaluated in vitro and antitumor efficacy was investigated in vivo as a single agent and in combination with irinotecan hydrochloride (CPT-11). The fusion protein RGD-L-TRAIL, but not TRAIL or RGE-L-TRAIL, specifically bound to microvascular endothelial cells in a dose-dependent manner and showed enhanced apoptosis-inducing activity (caspase-3 and caspase-8 activation) in alpha(V)beta(3) and alpha(V)beta(5) integrin-positive cancer cells. In addition, RGD-L-TRAIL was more effective in suppressing tumor growth of COLO-205 tumor-bearing mice than an equivalent dose of TRAIL. The antitumor effect of RGD-L-TRAIL was further enhanced by combination with CPT-11 in both TRAIL-sensitive COLO-205 and TRAIL-resistive HT-29 tumor xenograft models. Our findings suggest that the novel fusion protein RGD-L-TRAIL can directly target tumor endothelial cells as well as alpha(V)beta(3) and alpha(V)beta(5) integrin-positive tumor cells. The tumor-targeted delivery of TRAIL derivatives, such as RGD-L-TRAIL, may prove to be a promising lead candidate for cancer therapy.

The Role of HOX Transcription Factors in Cancer Predisposition and Progression.

Homeobox (HOX) transcription factors, encoded by a subset of homeodomain superfamily genes, play pivotal roles in many aspects of cellular physiology, embryonic development, and tissue homeostasis. Findings over the past decade have revealed that mutations in HOX genes can lead to increased cancer predisposition, and HOX genes might mediate the effect of many other cancer susceptibility factors by recognizing or executing altered genetic information. Remarkably, several lines of evidence highlight the interplays between HOX transcription factors and cancer risk loci discovered by genome-wide association studies, thereby gaining molecular and biological insight into cancer etiology. In addition, deregulated HOX gene expression impacts various aspects of cancer progression, including tumor angiogenesis, cell autophagy, proliferation, apoptosis, tumor cell migration, and metabolism. In this review, we will discuss the fundamental roles of HOX genes in cancer susceptibility and progression, highlighting multiple molecular mechanisms of HOX involved gene misregulation, as well as their potential implications in clinical practice.

A qPCR method for genome editing efficiency determination and single-cell clone screening in human cells.

CRISPR/Cas9 technology has been widely used for targeted genome modification both in vivo and in vitro. However, an effective method for evaluating genome editing efficiency and screening single-cell clones for desired modification is still lacking. Here, we developed this real time PCR method based on the sensitivity of Taq DNA polymerase to nucleotide mismatch at primer 3' end during initiating DNA replication. Applications to CRISPR gRNAs targeting EMX1, DYRK1A and HOXB13 genes in Lenti-X 293 T cells exhibited comprehensive advantages. Just in one-round qPCR analysis using genomic DNA from cells underwent CRISPR/Cas9 or BE4 treatments, the genome editing efficiency could be determined accurately and quickly, for indel, HDR as well as base editing. When applied to single-cell clone screening, the genotype of each cell colony could also be determined accurately. This method defined a rigorous and practical way in quantify genome editing events.

Comparative profiling of analog targets: a case study on resveratrol for mouse melanoma metastasis suppression.

Many plant-specialized metabolites have remedial properties and provide an endless chemical resource for drug discovery. However, most of these metabolites have promiscuous binding targets in mammalian cells and elicit a series of responses that collectively change the physiology of the cells. To explore the potential of these multi-functional and multi-targeted drugs, it is critical to understand the direct relationships between their key chemical features, the corresponding binding targets and the relevant biological effects, which is a prerequisite for future drug modification and optimization. Methods: We introduced and demonstrated a general workflow, called Comparative Profiling of Analog Targets (CPAT), to connect specific biological effects with defined chemical structures of drugs. Using resveratrol (RSV) as an example, we have synthesized and characterized a series of partial functional analogs of RSV. An analog (named RSVN) that specifically lost the inhibitory effect of RSV in cell migration was identified. The binding targets of RSVN and RSV was profiled and compared. Results: Comparative profiling of the RSV and RSVN binding targets showed that, unlike RSV, RSVN failed to target specific components involved in DNA methylation (histone deacetylase 1 [HDAC1] and DNA methyltransferase 3 alpha [DNMT3a]), suggesting that RSV suppresses cell migration through epigenetic regulation. Indeed, RSV treatment recruited HDAC1 and DNMT3a to the promoter region of the focal adhesion kinase (FAK), a key factor involved in cell adhesion, enhanced the promoter methylation, and thus attenuated the protein expression. The inhibitory effect of RSV in cell migration was diminished once FAK expression was restored. Thus, the mechanism of RSV in inhibiting cell migration could be largely accounted to epigenetically control of FAK expression. Conclusion: Our results showed that even though RSV exhibits promiscuous binding, its inhibitory effect on cell migration can be mechanistically understood. First, the presence of 4'-hydroxystilbene within the RSV structure is essential for this activity. Second, it inhibits cell migration through epigenetically based downregulation of FAK expression. Taken together, we propose that CPAT might also be adapted to delineate the specific function of other natural products (NPs) that exhibit binding promiscuity.