Implications For Human Cancer Research Articles

A guide to selecting high-performing antibodies for Secreted frizzled-related protein 1 (sFRP-1) for use in Western Blot and immunoprecipitation

Secreted frizzled-related protein 1 (sFRP-1) is a secreted protein, belonging to the secreted glycoprotein SFRP family. As a modulator of the Wnt/β-catenin signalling pathway, sFRP-1 has implications in human cancers and neurological diseases. If the community had access to well-characterized anti-sFRP-1 antibodies, the reproducibility of sFRP-1 research would be enhanced. In this study, we characterized 11 sFRP-1 commercial antibodies for Western Blot and immunoprecipitation, using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address the antibody reproducibility issue by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.

The identification of high-performing antibodies for Secreted frizzled-related protein 1 (sFRP-1) for use in Western Blot and immunoprecipitation

Secreted frizzled-related protein 1 (sFRP-1) is a secreted protein, belonging to the secreted glycoprotein SFRP family. As a modulator of the Wnt/β-catenin signalling pathway, sFRP-1 has implications in human cancers and neurologic diseases. If the community had access to well-characterized anti-sFRP-1 antibodies, the reproducibility of sFRP-1 research would be enhanced. In this study, we characterized 11 sFRP-1 commercial antibodies for Western Blot and immunoprecipitation, using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified many well-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.

Definition of a Novel Cuproptosis-Relevant lncRNA Signature for Uncovering Distinct Survival, Genomic Alterations, and Treatment Implications in Lung Adenocarcinoma.

Objective Cuproptosis is a newly discovered copper-independent cell death modality, and limited evidence suggests the critical implications in human cancers. Nonetheless, the clinical impacts of cuproptosis-relevant lncRNAs in lung adenocarcinoma (LUAD) remain largely ill-defined. The present study was aimed at defining a cuproptosis-relevant lncRNA signature for LUAD and discuss the clinical utility. Methods We collected transcriptome expression profiling, clinical information, somatic mutation, and copy number variations from TCGA-LUAD cohort retrospectively. The genetic alterations of cuproptosis genes were systematically assessed across LUAD, and cuproptosis-relevant lncRNAs were screened for defining a LASSO prognostic model. Genomic alterations, immunological and stemness features, and therapeutic sensitivity were studied with a series of computational approaches. Results Cuproptosis genes displayed aberrant expression and widespread genomic alterations across LUAD, potentially modulated by m6A/m5C/m1A RNA modification mechanisms. We defined a cuproptosis-relevant lncRNA signature, with a reliable efficacy in predicting clinical outcomes. High-risk subset displayed higher somatic mutations, CNVs, TMB, SNV neoantigens, aneuploidy score, CTA score, homologous recombination defects, and intratumor heterogeneity, cytolytic activity, CD8+ T effector, and antigen processing machinery, proving that this subset might benefit from immunotherapy. Increased stemness indexes and activity of oncogenic pathways might contribute to undesirable prognostic outcomes for high-risk subset. Additionally, high-risk patients generally exhibited higher response to chemotherapeutic agents (cisplatin, etc.). We also predicted several small molecule compounds (GSK461364, KX2-391, etc.) for treating this subset. Conclusion Accordingly, this cuproptosis-relevant lncRNA signature offers an efficient approach to identify and characterize diverse prognosis, genomic alterations, and treatment outcomes in LUAD, thus potentially assisting personalized therapy.

Comprehensive characterization genetic regulation and chromatin landscape of enhancer-associated long non-coding RNAs and their implication in human cancer.

Long non-coding RNAs (lncRNAs) that emanate from enhancer regions (defined as enhancer-associated lncRNAs, or elncRNAs) are emerging as critical regulators in disease progression. However, their biological characteristics and clinical relevance have not been fully portrayed. Here, based on the traditional expression quantitative loci (eQTL) and our optimized residual eQTL method, we comprehensively described the genetic effect on elncRNA expression in more than 300 lymphoblastoid cell lines. Meanwhile, a chromatin atlas of elncRNAs relative to the genetic regulation state was depicted. By applying the maximum likelihood estimate method, we successfully identified causal elncRNAs for protein-coding gene expression reprogramming and showed their associated single nucleotide polymorphisms (SNPs) favor binding of transcription factors. Further epigenome analysis revealed two immune-associated elncRNAs AL662844.4 and LINC01215 possess high levels of H3K27ac and H3K4me1 in human cancer. Besides, pan-cancer analysis of 3D genome, transcriptome, and regulatome data showed they potentially regulate tumor-immune cell interaction through affecting MHC class I genes and CD47, respectively. Moreover, our study showed there exist associations between elncRNA and patient survival. Finally, we made a user-friendly web interface available for exploring the regulatory relationship of SNP-elncRNA-protein-coding gene triplets (http://bio-bigdata.hrbmu.edu.cn/elncVarReg). Our study provides critical mechanistic insights for elncRNA function and illustrates their implications in human cancer.

The MLL3/4 H3K4 methyltransferase complex in establishing an active enhancer landscape.

Enhancers are cis-regulatory elements that play essential roles in tissue-specific gene expression during development. Enhancer function in the expression of developmental genes requires precise regulation, while deregulation of enhancer function could be the main cause of tissue-specific cancer development. MLL3/KMT2C and MLL4/KMT2D are two paralogous histone modifiers that belong to the SET1/MLL (also named COMPASS) family of lysine methyltransferases and play critical roles in enhancer-regulated gene activation. Importantly, large-scale DNA sequencing studies have revealed that they are amongst the most frequently mutated genes associated with human cancers. MLL3 and MLL4 form identical multi-protein complexes for modifying mono-methylation of histone H3 lysine 4 (H3K4) at enhancers, which together with the p300/CBP-mediated H3K27 acetylation can generate an active enhancer landscape for long-range target gene activation. Recent studies have provided a better understanding of the possible mechanisms underlying the roles of MLL3/MLL4 complexes in enhancer regulation. Moreover, accumulating studies offer new insights into our knowledge of the potential role of MLL3/MLL4 in cancer development. In this review, we summarize recent evidence on the molecular mechanisms of MLL3/MLL4 in the regulation of active enhancer landscape and long-range gene expression, and discuss their clinical implications in human cancers.

Circular RNA hsa_circ_0061825 (circ-TFF1) contributes to breast cancer progression through targeting miR-326/TFF1 signalling.

ObjectivesCircular RNAs (circRNAs) are RNA transcripts that belong to non‐coding RNAs (ncRNAs), whose implication in human cancers has been recently demonstrated. However, the specific role of multiple circRNAs in breast cancer remains unidentified.Materials and methodsMicroarray analysis and bioinformatics analysis were applied to select circRNA and miRNA, respectively. The loop structure of circ‐TFF1 was confirmed using RNase R treatment, divergent primer PCR and Sanger sequencing. qRT‐PCR and Western blot were employed for gene expressions. In vitro and in vivo experiments were conducted to assess the function of circ‐TFF1 in biological processes in breast cancer cells. FISH and subcellular separation indicated circ‐TFF1 cellular distribution. Luciferase reporter and RIP assays and Pearson's correlation analysis were performed to evaluate relationships between genes.ResultsCirc‐TFF1 and TFF1 were both upregulated and positively associated with each other in breast cancer. Knockdown of circ‐TFF1 hindered breast cancer cell proliferation, migration, invasion and EMT in vitro and controlled tumour growth in vivo. Circ‐TFF1 acted as a ceRNA of TFF1 by sponging miR‐326, and its contribution to breast cancer progression was mediated by miR‐326/TFF1 axis.ConclusionsCirc‐TFF1 is a facilitator in breast cancer relying on TFF1 by absorbing miR‐326, providing a novel promising target for BC treatment.

Cullin RING Ligase 5 (CRL-5): Neddylation Activation and Biological Functions.

Cullin-5 (Cul-5) was originally identified as an arginine vasopressin (AVP) receptor due to its homology to a vasopressin-activated calcium-mobilizing protein 1 (VACM-1). Cul-5 has subsequently gained much attention after being identified as the key component of CRL-5 (Cullin-RING ligase-5) that mediates ubiquitylation and degradation of several key cellular proteins associated with human cancers and viral infections. Structurally, Cul-5 interacts with the Elongin B/C complex, a RING finger protein (RBX2/SAG), and a SOCS protein to form a CRL-5 E3 ubiquitin ligase protein complex. CRL-5, by controlling turnover of a variety of substrates, is implicated in several biological processes and human diseases. Activation of CRL-5 requires Cul-5 neddylation, catalyzed by a neddylation enzyme cascade, consisting of the E1 NEDD8-activating enzyme (NAE), the E2 neddylation conjugating enzyme (UBE2F), and E3 neddylation ligase (RBX2/SAG). RBX2/SAG, therefore, serves as both Cul-5 neddylation E3 and CRL-5 ubiquitylation E3. Here, we review the current knowledge on CRL-5, its activation by the UBE2F-SAG, its regulation of various signaling pathways via substrate degradation, and its implications in human cancers.

Interferon regulatory factor 1 inactivation in human cancer.

Interferon regulatory factors (IRFs) are a group of closely related proteins collectively referred to as the IRF family. Members of this family were originally recognized for their roles in inflammatory responses; however, recent research has suggested that they are also involved in tumor biology. This review focusses on current knowledge of the roles of IRF-1 and IRF-2 in human cancer, with particular attention paid to the impact of IRF-1 inactivation. The different mechanisms underlying IRF-1 inactivation and their implications for human cancers and the potential importance of IRF-1 in immunotherapy are also summarized.

Xom, a ventralizing factor, regulates beta-catenin levels and cell fate.

Read the Original article at doi:10.1002/1873-3468.12949

Mechanistic regulation of epithelial-to-mesenchymal transition through RAS signaling pathway and therapeutic implications in human cancer.

RAS effector signaling instead of being simple, unidirectional and linear cascade, is actually recognized as highly complex and dynamic signaling network. RAF-MEK-ERK cascade, being at the center of complex signaling network, links to multiple scaffold proteins through feed forward and feedback mechanisms and dynamically regulate tumor initiation and progression. Three isoforms of Ras harbor mutations in a cell and tissue specific manner. Besides mutations, their epigenetic silencing also attributes them to exhibit oncogenic activities. Recent evidences support the functions of RAS oncoproteins in the acquisition of tumor cells with Epithelial-to-mesenchymal transition (EMT) features/ epithelial plasticity, enhanced metastatic potential and poor patient survival. Google Scholar electronic databases and PubMed were searched for original papers and reviews available till date to collect information on stimulation of EMT core inducers in a Ras driven cancer and their regulation in metastatic spread. Improved understanding of the mechanistic basis of regulatory interactions of microRNAs (miRs) and EMT by reprogramming the expression of targets in Ras activated cancer, may help in designing effective anticancer therapies. Apparent lack of adverse events associated with the delivery of miRs and tissue response make 'drug target miRNA' an ideal therapeutic tool to achieve progression free clinical response.

Dynamic and Modularized MicroRNA Regulation and Its Implication in Human Cancers

MicroRNA is responsible for the fine-tuning of fundamental cellular activities and human disease development. The altered availability of microRNAs, target mRNAs, and other types of endogenous RNAs competing for microRNA interactions reflects the dynamic and conditional property of microRNA-mediated gene regulation that remains under-investigated. Here we propose a new integrative method to study this dynamic process by considering both competing and cooperative mechanisms and identifying functional modules where different microRNAs co-regulate the same functional process. Specifically, a new pipeline was built based on a meta-Lasso regression model and the proof-of-concept study was performed using a large-scale genomic dataset from ~4,200 patients with 9 cancer types. In the analysis, 10,726 microRNA-mRNA interactions were identified to be associated with a specific stage and/or type of cancer, which demonstrated the dynamic and conditional miRNA regulation during cancer progression. On the other hands, we detected 4,134 regulatory modules that exhibit high fidelity of microRNA function through selective microRNA-mRNA binding and modulation. For example, miR-18a-3p, −320a, −193b-3p, and −92b-3p co-regulate the glycolysis/gluconeogenesis and focal adhesion in cancers of kidney, liver, lung, and uterus. Furthermore, several new insights into dynamic microRNA regulation in cancers have been discovered in this study.

Biology and insights into the role of cohesin protease separase in human malignancies.

Separase, an enzyme that resolves sister chromatid cohesion during the metaphase-to-anaphase transition, plays a pivotal role in chromosomal segregation and cell division. Separase protein, encoded by the extra spindle pole bodies like 1 (ESPL1) gene, is overexpressed in numerous human cancers including breast, bone, brain, and prostate. Separase is oncogenic, and its overexpression is sufficient to induce mammary tumours in mice. Either acute or chronic overexpression of separase in mouse mammary glands leads to aneuploidy and tumorigenesis, and inhibition of separase enzymatic activity decreases the growth of human breast tumour xenografts in mice. This review focuses on the biology of and insights into the molecular mechanisms of separase as an oncogene, and its significance and implications for human cancers.

An Evolution-Guided Analysis Reveals a Multi-Signaling Regulation of Fas by Tyrosine Phosphorylation and its Implication in Human Cancers.

Demonstrations of both pro-apoptotic and pro-survival abilities of Fas (TNFRSF6/CD95/APO-1) have led to a shift from the exclusive “Fas apoptosis” to “Fas multisignals” paradigm and the acceptance that Fas-related therapies face a major challenge, as it remains unclear what determines the mode of Fas signaling. Through protein evolution analysis, which reveals unconventional substitutions of Fas tyrosine during divergent evolution, evolution-guided tyrosine-phosphorylated Fas proxy, and site-specific phosphorylation detection, we show that the Fas signaling outcome is determined by the tyrosine phosphorylation status of its death domain. The phosphorylation dominantly turns off the Fas-mediated apoptotic signal, while turning on the pro-survival signal. We show that while phosphorylations at Y232 and Y291 share some common functions, their contributions to Fas signaling differ at several levels. The findings that Fas tyrosine phosphorylation is regulated by Src family kinases (SFKs) and the phosphatase SHP-1 and that Y291 phosphorylation primes clathrin-dependent Fas endocytosis, which contributes to Fas pro-survival signaling, reveals for the first time the mechanistic link between SFK/SHP-1-dependent Fas tyrosine phosphorylation, internalization route, and signaling choice. We also demonstrate that levels of phosphorylated Y232 and Y291 differ among human cancer types and differentially respond to anticancer therapy, suggesting context-dependent involvement of Fas phosphorylation in cancer. This report provides a new insight into the control of TNF receptor multisignaling by receptor phosphorylation and its implication in cancer biology, which brings us a step closer to overcoming the challenge in handling Fas signaling in treatments of cancer as well as other pathologies such as autoimmune and degenerative diseases.

Methylation-dependent regulation of HIF-1α stability restricts retinal and tumour angiogenesis.

Hypoxia-inducible factor-1α (HIF-1α) mediates hypoxic responses and regulates gene expression involved in angiogenesis, invasion and metabolism. Among the various HIF-1α posttranslational modifications, HIF-1α methylation and its physiological role have not yet been elucidated. Here we show that HIF-1α is methylated by SET7/9 methyltransferase, and that lysine-specific demethylase 1 reverses its methylation. The functional consequence of HIF-1α methylation is the modulation of HIF-1α stability primarily in the nucleus, independent of its proline hydroxylation, during long-term hypoxic and normoxic conditions. Knock-in mice bearing a methylation-defective Hif1aKA/KA allele exhibit enhanced retinal angiogenesis and tumour vascularization via HIF-1α stabilization. Importantly, S28Y and R30Q mutations of HIF-1α, found in human cancers, are involved in the altered HIF-1α stability. Together, these results demonstrate a role for HIF-1α methylation in regulating protein stability, thereby modulating biological output including retinal and tumour angiogenesis, with therapeutic implications in human cancer.

Genetic and epigenetic alterations of microRNAs and implications for human cancers and other diseases.

MicroRNAs (miRNAs) are a well-studied group of noncoding RNAs that control gene expression by interacting mainly with messenger RNA. It is known that miRNAs and their biogenesis regulatory machineries have crucial roles in multiple cell processes; thus, alterations in these genes often lead to disease, such as cancer. Disruption of these genes can occur through epigenetic and genetic alterations, resulting in aberrant expression of miRNAs and subsequently of their target genes. This review focuses on the disruption of miRNAs and their key regulatory machineries by genetic alterations, with emphasis on mutations and epigenetic changes in cancer and other diseases.

Combinatorial Control of mRNA Fates by RNA-Binding Proteins and Non-Coding RNAs

Post-transcriptional control of gene expression is mediated by RNA-binding proteins (RBPs) and small non-coding RNAs (e.g., microRNAs) that bind to distinct elements in their mRNA targets. Here, we review recent examples describing the synergistic and/or antagonistic effects mediated by RBPs and miRNAs to determine the localisation, stability and translation of mRNAs in mammalian cells. From these studies, it is becoming increasingly apparent that dynamic rearrangements of RNA-protein complexes could have profound implications in human cancer, in synaptic plasticity, and in cellular differentiation.

Varying the Terrain of Epigenetic Landscapes: Implications for Human Cancer

The journey from embryonic stem cell to a fully developed liver, heart or muscle cell requires not only having the right set of genes, but also having genes that are turned on and off at the right time — a job that is handled in part by DNA‐packaging proteins known as histones.Cells use a number of mechanisms to establish and maintain the activation or silencing of specific genes. Among these is the chemical modification of histones, discussed in the second lecture. In addition, histone variants, which differ from other histone proteins by just a handful of amino acids, can be inserted at specific locations in the genome to provide a cell with another mechanism for fine‐tuning gene regulation. Dr. Allis' group has shown that histone variants play an important role in determining how and when genes are read or are silenced.Recent work from Dr. Allis' laboratory has focused upon one member of the histone H3 family known as H3.3. In comparing the genome‐wide localization of H3.3, Allis and co‐workers found that histone H3.3 is prevalent in regions of the genome where active genes are found, as well as in silent regions, such as at the ends of chromosomes, called telomeres. They went on to identify several additional proteins associated with H3.3. Two of them, ATRX and Daxx, were then discovered to be frequently mutated in a sporadic, non‐functional pancreatic cancers, known as panNETs. Current experiments seek to determine whether genetic deficiency of Daxx and ATRX in pancreatic neuroendocrine cells is sufficient to induce tumorigenesis.Mutations in histone H3.3 itself have also been found in pediatric brain tumors (gliomas). Allis and co‐workers have recently asked ‐‐ how one of these mutations might act cause cancer in young children‾ Surprisingly they find that a specific mutation in H3.3 often found in the pediatric brain tumors inhibits histone methylation of all histone H3 at this site, a site methylated to induce gene silencing. Moreover, the specific mutation in H3.3 acts by inhibiting the enzyme responsible for bringing about methylation at this site. These data indicate that this specific type of mutation in histones might be more broadly applicable to the development and progression of other tumour types. It is not yet clear why aberrant epigenetic silencing of this enzyme results in the development of pediatric brain tumors, and further research is needed in an attempt to develop a better understanding of these difficult to treat childhood cancers.

Interaction of autophagy with microRNAs and their potential therapeutic implications in human cancers

Autophagy is a tightly regulated intracellular self-digestive process involving the lysosomal degradation of cytoplasmic organelles and proteins. A number of studies have shown that autophagy is dysregulated in cancer initiation and progression, or cancer cells under various stress conditions. As a catabolic pathway conserved among eukaryotes, autophagy is regulated by the autophagy related genes and pathways. MicroRNAs (miRNAs) are small, non-coding endogenous RNAs that may regulate almost every cellular process including autophagy. And autophagy is also involved in the regulation of miRNAs expression and homeostasis. Here we reviewed some literatures on the interaction of miRNAs with autophagy and the application of miRNAs-mediated autophagic networks as a promising target in pre-clinical cancer models. Furthermore, strategies of miRNAs delivery for miRNAs-based anti-cancer therapy will also be summarized and discussed.

The methyltransferase G9a regulates HoxA9-dependent transcription in AML

Chromatin modulators are emerging as attractive drug targets, given their widespread implication in human cancers and susceptibility to pharmacological inhibition. Here we establish the histone methyltransferase G9a/EHMT2 as a selective regulator of fast proliferating myeloid progenitors with no discernible function in hematopoietic stem cells (HSCs). In mouse models of acute myeloid leukemia (AML), loss of G9a significantly delays disease progression and reduces leukemia stem cell (LSC) frequency. We connect this function of G9a to its methyltransferase activity and its interaction with the leukemogenic transcription factor HoxA9 and provide evidence that primary human AML cells are sensitive to G9A inhibition. Our results highlight a clinical potential of G9A inhibition as a means to counteract the proliferation and self-renewal of AML cells by attenuating HoxA9-dependent transcription.

Ectopic γ-catenin Expression Partially Mimics the Effects of Stabilized β-catenin on Embryonic Stem Cell Differentiation

β-catenin, an adherens junction component and key Wnt pathway effector, regulates numerous developmental processes and supports embryonic stem cell (ESC) pluripotency in specific contexts. The β-catenin homologue γ-catenin (also known as Plakoglobin) is a constituent of desmosomes and adherens junctions and may participate in Wnt signaling in certain situations. Here, we use β-catenin(+/+) and β-catenin(−/−) mouse embryonic stem cells (mESCs) to investigate the role of γ-catenin in Wnt signaling and mESC differentiation. Although γ-catenin protein is markedly stabilized upon inhibition or ablation of GSK-3 in wild-type (WT) mESCs, efficient silencing of its expression in these cells does not affect β-catenin/TCF target gene activation after Wnt pathway stimulation. Nonetheless, knocking down γ-catenin expression in WT mESCs appears to promote their exit from pluripotency in short-term differentiation assays. In β-catenin(−/−) mESCs, GSK-3 inhibition does not detectably alter cytosolic γ-catenin levels and does not activate TCF target genes. Intriguingly, β-catenin/TCF target genes are induced in β-catenin(−/−) mESCs overexpressing stabilized γ-catenin and the ability of these genes to be activated upon GSK-3 inhibition is partially restored when wild-type γ-catenin is overexpressed in these cells. This suggests that a critical threshold level of total catenin expression must be attained before there is sufficient signaling-competent γ-catenin available to respond to GSK-3 inhibition and to regulate target genes as a consequence. WT mESCs stably overexpressing γ-catenin exhibit robust Wnt pathway activation and display a block in tri-lineage differentiation that largely mimics that observed upon overexpression of β-catenin. However, β-catenin overexpression appears to be more effective than γ-catenin overexpression in sustaining the retention of markers of naïve pluripotency in cells that have been subjected to differentiation-inducing conditions. Collectively, our study reveals a function for γ-catenin in the regulation of mESC differentiation and has implications for human cancers in which γ-catenin is mutated and/or aberrantly expressed.