Novel WRN Helicase Inhibitors Selectively Target Microsatellite Unstable Cancer Cells.

Microsatellite-unstable (MSI) cancers require WRN helicase to resolve replication stress due to expanded DNA (TA)n-dinucleotide repeats. WRN is a promising synthetic lethal target for MSI tumours, and WRN inhibitors are in development. Here, we used CRISPR-Cas9 base editing to map WRN residues critical for MSI cells, validating the helicase domain as the primary drug target. Fragment-based screening led to the development of potent and highly selective WRN helicase covalent inhibitors. These compounds selectively suppressed MSI model growth In vitro and In vivo by mimicking WRN loss, inducing DNA double-strand breaks at expanded TA-repeats and DNA damage. Assessment of biomarkers in preclinical models linked TA-repeat expansions and mismatch repair (MMR) alterations to compound activity. Efficacy was confirmed in immunotherapy-resistant organoids and patient-derived xenograft (PDX) models. The discovery of potent, selective covalent WRN inhibitors provides proof of concept for synthetic-lethal targeting of WRN in MSI cancer and tools to dissect WRN biology.

Prioritization of cancer therapeutic targets using CRISPR-Cas9 screens.

Functional genomics approaches can overcome limitations-such as the lack of identification of robust targets and poor clinical efficacy-that hamper cancer drug development. Here we performed genome-scale CRISPR-Cas9 screens in 324 human cancer cell lines from 30 cancer types and developed a data-driven framework to prioritize candidates for cancer therapeutics. We integrated cell fitness effects with genomic biomarkers and target tractability for drug development to systematically prioritize new targets in defined tissues and genotypes. We verified one of our most promising dependencies, the Werner syndrome ATP-dependent helicase, as a synthetic lethal target in tumours from multiple cancer types with microsatellite instability. Our analysis provides a resource of cancer dependencies, generates a framework to prioritize cancer drug targets and suggests specific new targets. The principles described in this study can inform the initial stages of drug development by contributing to a new, diverse and more effective portfolio of cancer drug targets.

Strategies for controlling CRISPR/Cas9 off-target effects and biological variations in mammalian genome editing experiments.

The CRISPR/Cas9 system has enabled efficient modification of genes in a variety of cellular systems for studying phenotypic effects of genetic perturbations. However, with this technology comes the inherent risk of generating off-target effects (OTEs) in addition to the desired modifications. As such, it can be difficult to conclusively determine that the observed phenotypic changes are in fact due to the intended modification of the target gene and not from random mutations elsewhere in the genome. In addition, biological variations observed within cultured cells or laboratory animals can also confound results and need to be addressed. In this article, we review potential sources of experimental and biological variation as well as propose experimental options to minimize and control OTEs and other variations in CRISPR genome editing experiments for exploratory research applications. Confirmation of on-target KO effect by orthogonal approaches is also discussed.

A Mec17-Myosin II Effector Axis Coordinates Microtubule Acetylation and Actin Dynamics to Control Primary Cilium Biogenesis.

Primary cilia are specialized, acetylated microtubule-based signaling processes. Cilium assembly is activated by cellular quiescence and requires reconfiguration of microtubules, the actin cytoskeleton, and vesicular trafficking machinery. How these components are coordinated to activate ciliogenesis remains unknown. Here we identify the microtubule acetyltransferase Mec-17 and myosin II motors as the key effectors in primary cilium biogenesis. We found that myosin IIB (Myh10) is required for cilium formation; however, myosin IIA (Myh9) suppresses it. Myh10 binds and antagonizes Myh9 to increase actin dynamics, which facilitates the assembly of the pericentrosomal preciliary complex (PPC) that supplies materials for cilium growth. Importantly, Myh10 expression is upregulated by serum-starvation and this induction requires Mec-17, which is itself accumulated upon cellular quiescence. Pharmacological stimulation of microtubule acetylation also induces Myh10 expression and cilium formation. Thus cellular quiescence induces Mec17 to couple the production of acetylated microtubules and Myh10, whose accumulation overcomes the inhibitory role of Myh9 and initiates ciliogenesis.

miR-33a promotes glioma-initiating cell self-renewal via PKA and NOTCH pathways.

Glioblastoma (GBM) is the most common and lethal brain tumor in adults. Glioma-initiating cells (GICs) are stem-like cells that have been implicated in glioblastoma progression and recurrence; however, the distinct properties of GICs and non-GICs within GBM tumors are largely uncharacterized. Here, we evaluated stem cell-associated microRNA (miR) expression in GICs from GBM patients and GICs derived from xenografted human glioma cell lines and determined that miR-33a promotes GIC growth and self-renewal. Moreover, evaluation of a GBM tissue array revealed that higher miR-33a expression was associated with poor prognosis of GBM patients. Antagonizing miR-33a function in GICs reduced self-renewal and tumor progression in immune-compromised mice, whereas overexpression of miR-33a in non-GICs promoted the display of features associated with GICs. We identified the mRNAs encoding phosphodiesterase 8A (PDE8A) and UV radiation resistance-associated gene (UVRAG) as direct miR-33a targets. PDE8A and UVRAG negatively regulated the cAMP/PKA and NOTCH pathways, respectively; therefore, miR-33a-dependent reduction of these proteins promoted growth and self-renewal of GICs by enhancing PKA and NOTCH activity. Furthermore, in GBM specimens, there was an inverse correlation between the expression levels of miR-33a and PDE8A and UVRAG expression. These findings reveal a miR-33a-centered signaling network that promotes GIC maintenance and has potential as a therapeutic target for GBM treatment.

Glycolysis-dependent histone deacetylase 4 degradation regulates inflammatory cytokine production.

Activation of the inflammatory response is accompanied by a metabolic shift to aerobic glycolysis. Here we identify histone deacetylase 4 (HDAC4) as a new component of the immunometabolic program. We show that HDAC4 is required for efficient inflammatory cytokine production activated by lipopolysaccharide (LPS). Surprisingly, prolonged LPS treatment leads to HDAC4 degradation. LPS-induced HDAC4 degradation requires active glycolysis controlled by GSK3ß and inducible nitric oxide synthase (iNOS). Inhibition of GSK3ß or iNOS suppresses nitric oxide (NO) production, glycolysis, and HDAC4 degradation. We present evidence that sustained glycolysis induced by LPS treatment activates caspase-3, which cleaves HDAC4 and triggers its degradation. Of importance, a caspase-3-resistant mutant HDAC4 escapes LPS-induced degradation and prolongs inflammatory cytokine production. Our findings identify the GSK3ß-iNOS-NO axis as a critical signaling cascade that couples inflammation to metabolic reprogramming and a glycolysis-driven negative feedback mechanism that limits inflammatory response by triggering HDAC4 degradation.

Association between soy isoflavone intake and breast cancer risk for pre- and post-menopausal women: a meta-analysis of epidemiological studies.

BACKGROUND: Conclusions drawn from meta-analyses on the association between soy isoflavone intake and breast cancer risk for pre- and post-menopausal women are not fully consistent. These meta-analyses did not explore the influence of different study designs on the pooled results on the basis of distinguishing between pre- and post-menopausal women. METHODOLOGY AND PRINCIPAL FINDINGS: We performed a meta-analysis of 35 studies which reported results of association between soy isoflavone intake and breast cancer risk for pre- and/or post-menopausal women, calculated pooled odds ratios and their 95% confidence intervals of pre- and post-menopausal women respectively, and further explored soy isoflavone-breast cancer association on the basis of considering different study regions and designs. Summary results suggested that soy isoflavone intake has a protective effect against breast cancer for both pre- and post-menopausal women. However, they are influenced by study design and region. Pooled ORs of studies carried out in Asian countries suggested that soy isoflavone's protective effect exist in both pre- and post-menopausal women (OR = 0.59, 95%CI: 0.48-0.69 for premenopausal women; OR = 0.59, 95%CI: 0.44-0.74 for postmenopausal women). However, there are some differences between the results pooled from different study designs for women in Asian countries (test for consistency, P = 0.04). Pooled OR of studies on postmenopausal women in Western countries suggested that soy isoflavone intake has a marginally significant protective effect (OR = 0.92; 95%CI: 0.83 ∼ 1.00), but further analyses stratifying by study design found no statistically significant association. CONCLUSIONS: We meta-analyzed more and newer research results, and separated women according to menopausal status to explore soy isoflavone-breast cancer association. We founded that soy isoflavone intake could lower the risk of breast cancer for both pre- and post-menopausal women in Asian countries. However, for women in Western countries, pre- or post-menopausal, there is no evidence to suggest an association between intake of soy isoflavone and breast cancer.

Microtubule acetylation amplifies p38 kinase signalling and anti-inflammatory IL-10 production.

Reversible acetylation of α-tubulin is an evolutionarily conserved modification in microtubule networks. Despite its prevalence, the physiological function and regulation of microtubule acetylation remain poorly understood. Here we report that macrophages challenged by bacterial lipopolysaccharides (LPS) undergo extensive microtubule acetylation. Suppression of LPS-induced microtubule acetylation by inactivating the tubulin acetyltransferase, MEC17, profoundly inhibits the induction of anti-inflammatory interleukin-10 (IL-10), a phenotype effectively reversed by an acetylation-mimicking α-tubulin mutant. Conversely, elevating microtubule acetylation by inhibiting the tubulin deacetylase, HDAC6, or stabilizing microtubules via Taxol stimulates IL-10 hyper-induction. Supporting the anti-inflammatory function of microtubule acetylation, HDAC6 inhibition significantly protects mice from LPS toxicity. In HDAC6-deficient macrophages challenged by LPS, p38 kinase signalling becomes selectively amplified, leading to SP1-dependent IL-10 transcription. Remarkably, the augmented p38 signalling is suppressed by MEC17 inactivation. Our findings identify reversible microtubule acetylation as a kinase signalling modulator and a key component in the inflammatory response.

Proteasomes activate aggresome disassembly and clearance by producing unanchored ubiquitin chains.

Aberrant protein aggregation is a dominant pathological feature in neurodegenerative diseases. Protein aggregates cannot be processed by the proteasome; instead, they are frequently concentrated to the aggresome, a perinuclear inclusion body, and subsequently removed by autophagy. Paradoxically, proteasomes are also concentrated at aggresomes and other related inclusion bodies prevalent in neurodegenerative disease. Here, we show that proteasomes are crucial components in aggresome clearance. The disassembly and disposal of aggresomes requires Poh1, a proteasomal deubiquitinating enzyme that cleaves ubiquitinated proteins and releases ubiquitin chains. In Poh1-deficient cells, aggresome clearance is blocked. Remarkably, microinjection of free lysine (K) 63-linked ubiquitin chains restores aggresome degradation. We present evidence that free ubiquitin chains produced by Poh1 bind and activate the deacetylase HDAC6, which, in turn, stimulates actinomyosin- and autophagy-dependent aggresome processing. Thus, unanchored ubiquitin chains are key signaling molecules that connect and coordinate the proteasome and autophagy to eliminate toxic protein aggregates.

Risk factors for breast cancer and expression of insulin-like growth factor-2 (IGF-2) in women with breast cancer in Wuhan City, China.

PURPOSE: The purpose of this study was to explore the risk factors for breast cancer and establish the expression rate of IGF-2 in female patients. METHODS: A case control study with 500 people in case group and 500 people in control group. A self-administered questionnaire was used to investigate risk factors for breast cancer. All cases were interviewed during a household survey. Immune-histochemical method was used to inspect the expression of IGF-2 in different tissues (benign breast lesions, breast cancer and tumor-adjacent tissue). RESULTS: Multivariate adjusted odds ratios and 95% confidence intervals were calculated using unconditional logistic regression. High body mass index (OR = 1.012,95%CI = 1.008-1.016), working attributes (OR = 1.004, 95%CI = 1.002 = 1.006), long menstrual period (OR = 1.007, 95%CI = 1.005-1.009), high parity OR = 1.003, 95%CI = 1.001-1.005) , frequent artificial abortion (OR = 1.004, 95%CI = 1.001-1.005), family history of cancer (OR = 1.003, 95%CI = 1.000-1.005), period of night shift (OR = 1.003, 95%CI = 1.001-1.006), live in high risk environment (OR = 1.005, 95%CI = 1.002-1.008), and family problems (OR = 1.010, 95%CI = 1.005-1.014) were associated with increased risk for breast cancer. In this study, good sleeping status, positive coping strategies, subjective support, and utility degree of social support were associated with reduced risk for breast cancer (OR = 0.998, 0.997, 0.985, 0.998 respectively; 95%CI = 0.996-1.000, 0.994-1.000, 0.980-0.989, 0.996-1.000, respectively). In benign breast lesions, breast cancer and tumor-adjacent tissue, IGF-2 was mainly expressed in the cytoplasm, but its expression rate was different (p<0.05). CONCLUSIONS: The incidence of breast cancer is a common result of multiple factors. IGF-2 is involved in the development of breast cancer, and its expression varies in different tissues (benign breast lesions, breast cancer and tumor-adjacent tissue).