CDKL3 is a targetable regulator of cell cycle progression in cancers.

Cell cycle regulation is largely abnormal in cancers. Molecular understanding and therapeutic targeting of the aberrant cell cycle are essential. Here, we identified that an underappreciated serine/threonine kinase, cyclin-dependent kinase-like 3 (CDKL3), crucially drives rapid cell cycle progression and cell growth in cancers. With regard to mechanism, CDKL3 localizes in the nucleus and associates with specific cyclin to directly phosphorylate retinoblastoma (Rb) for quiescence exit. In parallel, CDKL3 prevents the ubiquitin-proteasomal degradation of cyclin-dependent kinase 4 (CDK4) by direct phosphorylation on T172 to sustain G1 phase advancement. The crucial function of CDKL3 in cancers was demonstrated both in vitro and in vivo. We also designed, synthesized, and characterized a first-in-class CDKL3-specific inhibitor, HZ1. HZ1 exhibits greater potency than CDK4/6 inhibitor in pan-cancer treatment by causing cell cycle arrest and overcomes acquired resistance to CDK4/6 inhibitor. In particular, CDKL3 has significant clinical relevance in colon cancer, and the effectiveness of HZ1 was demonstrated by murine and patient-derived cancer models. Collectively, this work presents an integrated paradigm of cancer cell cycle regulation and suggests CDKL3 targeting as a feasible approach in cancer treatment.

Astrocyte-derived lactoferrin reduces ß-amyloid burden by promoting the interaction between p38 kinase and PP2A phosphatase in male APP/PS1 transgenic mice.

BACKGROUND AND PURPOSE: Overexpression of astrocytic lactoferrin (Lf) was observed in the brain of Alzheimer's disease (AD) patients, whereas the role of astrocytic Lf in AD progression remains unexplored. In this study, we aimed to evaluate the effects of astrocytic Lf on AD progression. EXPERIMENTAL APPROACH: Male APP/PS1 mice with astrocytes overexpressing human Lf were developed to evaluate the effects of astrocytic Lf on AD progression. N2a-sw cells also were employed to further uncover the mechanism of astrocytic Lf on ß-amyloid (Aß) production. KEY RESULTS: Astrocytic Lf overexpression increased protein phosphatase 2A (PP2A) activity and reduced amyloid precursor protein (APP) phosphorylation, Aß burden and tau hyperphosphorylation in APP/PS1 mice. Mechanistically, astrocytic Lf overexpression promoted the uptake of astrocytic Lf into neurons in APP/PS1 mice, and conditional medium from astrocytes overexpressing Lf inhibited p-APP (Thr668) expression in N2a-sw cells. Furthermore, recombinant human Lf (hLf) significantly enhanced PP2A activity and inhibited p-APP expression, whereas inhibition of p38 or PP2A activities abrogated the hLf-induced p-APP down-regulation in N2a-sw cells. Additionally, hLf promoted the interaction of p38 and PP2A via p38 activation, thereby enhancing PP2A activity, and low-density lipoprotein receptor-related protein 1 (LRP1) knockdown significantly reversed the hLf-induced p38 activation and p-APP down-regulation. CONCLUSIONS AND IMPLICATIONS: Our data suggested that astrocytic Lf promoted neuronal p38 activation, via targeting to LRP1, subsequently promoting p38 binding to PP2A to enhance PP2A enzyme activity, which finally inhibited Aß production via APP dephosphorylation. In conclusion, promoting astrocytic Lf expression may be a potential strategy against AD. LINKED ARTICLES: This article is part of a themed issue From Alzheimer's Disease to Vascular Dementia: Different Roads Leading to Cognitive Decline. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v181.6/issuetoc.

USP10 strikes down ß-catenin by dual-wielding deubiquitinase activity and phase separation potential.

Wnt/ß-catenin signaling is a conserved pathway crucially governing development, homeostasis, and oncogenesis. Discoveries of its regulators hold great values in both basic and translational research. Through screening, we identified a deubiquitinase, USP10, as a critical modulator of ß-catenin. Mechanistically, USP10 binds to key scaffold Axin1 via conserved motifs and stabilizes Axin1 through K48-linked deubiquitination. Surprisingly, USP10 physically tethers Axin1 and ß-catenin and promotes the phase separation for ß-catenin suppression regardless of the enzymatic activity. Function-wise, USP10 enzymatic activity preferably regulates embryonic development and both the enzymatic activity and physical function jointly control intestinal homeostasis by antagonizing ß-catenin. In colorectal cancer, USP10 substantially represses cancer growth mainly through physical promotion of phase separation and correlates with Wnt/ß-catenin magnitude clinically. Collectively, we discovered USP10 functioning in multiple biological processes against ß-catenin and unearthed the enzyme-dependent and -independent "dual-regulating" mechanism. These two functions of USP10 work in parallel and are context dependent.

Anti-Pan-Rspo Chimeric Protein-Conjugated Albumin Nanoparticle Provides Promising Opportunities in Cancer Targeted Therapy.

Rspos (R-spondins) belong to a family of secreted proteins that causes various cancers via interacting the corresponding receptors. However, targeted therapeutic approaches against Rspos are largely lacking. In this study, a chimeric protein Rspo-targeting anticancer chimeric protein (RTAC) is originally designed, engineered, and characterized. RTAC shows satisfactory anticancer effects through inhibition of pan-Rspo-mediated Wnt/ß-catenin signaling activation both in vitro and in vivo. Furthermore, a conceptually novel antitumor strategy distinct from traditional drug delivery systems that release drugs inside tumor cells is proposed. A special "firewall" nano-system is designed to enrich on tumor cell surface and cover the plasma membrane, rather than undergoing endocytosis, to block oncogenic Rspos from binding to receptors. Cyclic RGD (Arg-Gly-Asp) peptide-linked globular cluster serum albumin nanoparticles (SANP) are integrated as a vehicle for conjugating RTAC (SANP-RTAC/RGD) for tumor tissue targeting. These nanoparticles can adhere to the tumor cell surface and enable RTAC to locally capture free Rspos with high spatial efficiency and selectivity to antagonize cancer progression. Therefore, this approach offers a new nanomedical anticancer route and obtains the "dual-targeting" capability for effective tumor clearance and low potential toxicity. This study presents a proof-of-concept for anti-pan-Rspo therapy and a nanoparticle-integrated paradigm for targeted cancer treatment.

Astrocyte-specific loss of lactoferrin influences neuronal structure and function by interfering with cholesterol synthesis.

Growing evidence indicates that circulating lactoferrin (Lf) is implicated in peripheral cholesterol metabolism disorders. It has emerged that the distribution of Lf changes in astrocytes of aging brains and those exhibiting neurodegeneration; however, its physiological and/or pathological role remains unknown. Here, we demonstrate that astrocyte-specific knockout of Lf (designated cKO) led to decreased body weight and cognitive abnormalities during early life in mice. Accordingly, there was a reduction in neuronal outgrowth and synaptic structure in cKO mice. Importantly, Lf deficiency in the primary astrocytes led to decreased sterol regulatory element binding protein 2 (Srebp2) activation and cholesterol production, and cholesterol content in cKO mice and/or in astrocytes was restored by exogenous Lf or a Srebp2 agonist. Moreover, neuronal dendritic complexity and total dendritic length were decreased after culture with the culture medium of the primary astrocytes derived from cKO mice and that this decrease was reversed after cholesterol supplementation. Alternatively, these alterations were associated with an activation of AMP-activated protein kinase (AMPK) and inhibition of SREBP2 nuclear translocation. These data suggest that astrocytic Lf might directly or indirectly control in situ cholesterol synthesis, which may be implicated in neurodevelopment and several neurological diseases.

Aberrant Cholesterol Metabolism and Wnt/ß-Catenin Signaling Coalesce via Frizzled5 in Supporting Cancer Growth.

Frizzled (Fzd) proteins are Wnt receptors and play essential roles in development, homeostasis, and oncogenesis. How Wnt/Fzd signaling is coupled to physiological regulation remains unknown. Cholesterol is reported as a signaling molecule regulating morphogen such as Hedgehog signaling. Despite the elusiveness of the in-depth mechanism, it is well-established that pancreatic cancer specially requires abnormal cholesterol metabolism levels for growth. In this study, it is unexpectedly found that among ten Fzds, Fzd5 has a unique capacity to bind cholesterol specifically through its conserved extracellular linker region. Cholesterol-binding enables Fzd5 palmitoylation, which is indispensable for receptor maturation and trafficking to the plasma membrane. In Wnt-addicted pancreatic ductal adenocarcinoma (PDAC), cholesterol stimulates tumor growth via Fzd5-mediated Wnt/ß-catenin signaling. A natural oxysterol, 25-hydroxylsterol competes with cholesterol and inhibits Fzd5 maturation and Wnt signaling, thereby alleviating PDAC growth. This cholesterol-receptor interaction and ensuing receptor lipidation uncover a novel mechanism by which Fzd5 acts as a cholesterol sensor and pivotal connection coupling lipid metabolism to morphogen signaling. These findings further suggest that cholesterol-targeting may provide new therapeutic opportunities for treating Wnt-dependent cancers.

Vitamin D deficiency exacerbates Alzheimer-like pathologies by reducing antioxidant capacity.

Vitamin D (VD) deficiency is prevalent among aging people and Alzheimer's disease (AD) patients. However, the roles of VD deficiency in the pathology of AD remain largely unexplored. In this study, APP/PS1 mice were fed a VD-deficient diet for 13 weeks to evaluate the effects of VD deficiency on the learning and memory functions and the neuropathological characteristics of the mice. Our study revealed that VD deficiency accelerated cognitive impairment in the APP/PS1 mice. Mechanistic studies revealed that VD deficiency promoted glial activation and increased inflammatory factor secretion. Furthermore, VD deficiency increased the production and deposition of Aß by elevating the expression levels of amyloid precursor protein (APP) and ß-site APP cleavage enzyme 1 (BACE1). In addition, VD deficiency increased the phosphorylation of Tau at Thr181, Thr205 and Ser396 by increasing the activities of cyclin-dependent kinase 5 (CDK5) and glycogen synthase kinase 3α/ß (GSK3α/ß) and promoted synaptic dystrophy and neuronal loss. All these effects of VD deficiency may be ascribed to enhanced oxidative stress via the downregulation of superoxide dismutase 1 (SOD1), glutathione peroxidase 4 (GPx4) and cystine/glutamate exchanger (xCT). Taken together, our data suggest that VD deficiency exacerbates Alzheimer-like pathologies via promoting inflammatory stress, increasing Aß production and elevating Tau phosphorylation by decreasing antioxidant capacity in the brains of APP/PS1 mice. Hence, rescuing the VD status of AD patients should be taken into consideration during the treatment of AD.

α-Lipoic Acid Maintains Brain Glucose Metabolism via BDNF/TrkB/HIF-1α Signaling Pathway in P301S Mice.

The microtubule-associated protein tau is closely correlated with hypometabolism in Alzheimer's disease (AD). α-lipoic acid (LA), which is a naturally occurring cofactor in mitochondrial, has been shown to have properties that can inhibit the tau pathology and neuronal damage in our previous research. However, if LA affects glucose metabolism when it reverses tau pathology remains unclear, especially concerning the potential mechanism. Therefore, we make a further study using the P301S mouse model (a tauopathy and AD mouse model which overexpressing fibrillary tau) to gain a clear idea of the aforementioned problems. Here, we found chronic LA administration significantly increased glucose availability by elevating glucose transporter 3 (GLUT3), GLUT4, vascular endothelial growth factor (VEGF) protein and mRNA level, and heme oxygenase-1 (HO-1) protein level in P301S mouse brains. Meanwhile, we found that LA also promoted glycolysis by directly upregulating hexokinase (HK) activity, indirectly by increasing proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and DNA repair enzymes (OGG1/2 and MTH1). Further, we found the underlying mechanism of restored glucose metabolism might involve in the activation of brain-derived neurotrophic factor (BDNF)/tyrosine Kinase receptor B (TrkB)/hypoxia-inducible factor-1α (HIF-1α) signaling pathway by LA treatment.

Lactoferrin ameliorates dopaminergic neurodegeneration and motor deficits in MPTP-treated mice.

Brain iron accumulation is common in patients with Parkinson's disease (PD). Iron chelators have been investigated for their ability to prevent neurodegenerative diseases with features of iron overload. Given the non-trivial side effects of classical iron chelators, lactoferrin (Lf), a multifunctional iron-binding globular glycoprotein, was screened to identify novel neuroprotective pathways against dopaminergic neuronal impairment. We found that Lf substantially ameliorated PD-like motor dysfunction in the subacute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of PD. We further showed that Lf could alleviate MPTP-triggered apoptosis of DA neurons, neuroinflammation, and histological alterations. As expected, we also found that Lf suppressed MPTP-induced excessive iron accumulation and the upregulation of divalent metal transporter (DMT1) and transferrin receptor (TFR), which is the main intracellular iron regulation protein, and subsequently improved the activity of several antioxidant enzymes. We probed further and determined that the neuroprotection provided by Lf was involved in the upregulated levels of brain-derived neurotrophic factor (BDNF), hypoxia-inducible factor 1α (HIF-1α) and its downstream protein, accompanied by the activation of extracellular regulated protein kinases (ERK) and cAMP response element binding protein (CREB), as well as decreased phosphorylation of c-Jun N-terminal kinase (JNK) and mitogen activated protein kinase (MAPK)/P38 kinase in vitro and in vivo. Our findings suggest that Lf may be an alternative safe drug in ameliorating MPTP-induced brain abnormalities and movement disorder.

Evaluation of cell death pathways initiated by antitumor drugs melatonin and valproic acid in bladder cancer cells.

Effective drug combinations have the potential to strengthen therapeutic efficacy and combat drug resistance. Both melatonin and valproic acid (VPA) exhibit antitumor activities in various cancer cells. The aim of this study was to evaluate the cell death pathways initiated by anticancer combinatorial effects of melatonin and VPA in bladder cancer cells. The results demonstrated that the combination of melatonin and VPA leads to significant synergistic growth inhibition of UC3 bladder cancer cells. Gene expression studies revealed that cotreatment with melatonin and VPA triggered the up-regulation of certain genes related to apoptosis (TNFRSF10A and TNFRSF10B), autophagy (BECN, ATG3 and ATG5) and necrosis (MLKL, PARP-1 and RIPK1). The combinatorial treatment increased the expression of endoplasmic reticulum (ER)-stress-related genes ATF6, IRE1, EDEM1 and ERdj4. Cotreatment with melatonin and VPA enhanced the expression of E-cadherin, and decreased the expression of N-cadherin, Fibronectin, Snail and Slug. Furthermore, the Wnt pathway and Raf/MEK/ERK pathway were activated by combinatorial treatment. However, the effects on the expression of certain genes were not further enhanced in cells following combinatorial treatment in comparison to individual treatment of melatonin or VPA. In summary, these findings provided evidence that cotreatment with melatonin and VPA exerted increased cytotoxicity by regulating cell death pathways in UC3 bladder cancer cells, but the clinical significance of combinatorial treatment still needs to be further exploited.