Cdk5 Knockdown Research Articles

PI3K/Akt inhibition promotes AR activity and prostate cancer cell proliferation through p35-CDK5 modulation

Aberrant PI3K/Akt activation is linked to prostate cancer (PCa) malignancy, while androgen receptor (AR) is critical in early-stage PCa development. Investigating the interaction between these pathways is crucial for PCa malignancy. Our previous study demonstrated that p35-CDK5 mediates post-translational modifications of AR, STAT3, and p21CIP1, eventually promoting PCa cell growth. This study revealed the role of p35-CDK5 in between PI3K/Akt and AR by utilizing LNCaP and 22Rv1 cells. Through the TCGA database analysis, we observed a positive correlation between PTEN and p35 expression, implying a potential negative correlation between PI3K/Akt activation and p35-CDK5. Inhibiting PI3K/Akt with LY294002, Capivasertib (AZD5363), or using an inactive Akt mutant significantly increased p35 expression and subsequently enhanced AR stability and activation in PCa cells. On the other hand, CDK5-knockdown reversed these effects. The involvement of the β-catenin/Egr1-axis was observed in regulating PI3K/Akt inhibition and p35-CDK5 activation, implying a possible mechanistic connection. Importantly, CDK5 knockdown further reduced PI3K/Akt-inhibition-induced AR and cell viability maintenance, suggesting a compensatory role for CDK5-AR in maintaining cell viability under Akt inhibition. In conclusion, PI3K/Akt inhibition could trigger p35-CDK5-dependent AR activation and cell viability, highlighting p35-CDK5 as a critical link connecting PI3K/Akt inhibition to AR activation and pivotal in PCa cell resistance to PI3K/Akt blockade.

Neurogenic Effects of Inorganic Arsenic and Cdk5 Knockdown in Zebrafish Embryos: A Perspective on Modeling Autism.

The exact mechanisms of the development of autism, a multifactorial neurological disorder, are not clear. The pathophysiology of autism is complex, and investigations at the cellular and molecular levels are ongoing to provide clarity. Mutations in specific genes have been identified as risk factors for autism. The role of heavy metals in the pathogenesis of autism is subject to many studies and remains debatable. Although no exact neuronal phenotypes have been identified linked to autistic symptoms, overproduction and reduction of specific neurons have been implicated. A growing literature on generating genetic and non-genetic models of autism aims to help with understanding mechanistic studies that can explain the complexity of the disorder. Both genetic and non-genetic methods of zebrafish have been used to model autism. For several human autism risk genes, validated zebrafish mutant models have been generated. There is growing evidence indicating a potential link between autism and inorganic arsenic exposure. We have previously shown that inorganic arsenic induces supernumerary spinal motor neurons via Sonic hedgehog (Shh) signaling pathway, and Cdk5 knockdown causes an overproduction of cranial and spinal motor neurons in zebrafish. Here, in this review, we provide a perspective on what these findings of neurogenic phenotypes mean in terms of dysregulated pathways of motor neuron development and their applicability to understanding cellular and molecular underpinnings of autism.

Cdk5 Promotes Mitochondrial Fission via Drp1 Phosphorylation at S616 in Chronic Ethanol Exposure-Induced Cognitive Impairment.

Excessive alcohol consumption can lead to alterations in brain structure and function, even causing irreversible learning and memory disorders. The hippocampus is one of the most sensitive areas to alcohol neurotoxicity in the brain. Accumulating evidence indicates that mitochondrial dysfunction contributes to alcohol neurotoxicity. However, little is known about the underlying molecular mechanisms. In this study, we found that chronic exposure to ethanol caused abnormal mitochondrial fission/fusion and morphology by activating the mitochondrial fission protein dynamin-related protein 1 (Drp1) and upregulating Drp1 receptors, such as fission protein 1 (Fis1), mitochondrial dynamics protein of 49kDa (Mid49), and mitochondrial fission factor (Mff), combined with decreasing optic atrophy 1 (Opa1) and mitochondrial fusion protein mitofusin 1 (Mfn1) levels. In addition, mitochondrial division inhibitor 1 (mdivi-1) abrogated ethanol-induced mitochondrial dysfunction and improved hippocampal synapses and cognitive function in ethanol-exposed mice. Chronic ethanol exposure also resulted in cyclin-dependent kinase 5 (Cdk5) overactivation, as shown by the increase in the levels of Cdk5 and its activator P25 in the hippocampus. Furthermore, a Cdk5/P25 inhibitor (roscovitine) or Cdk5 knockdown using small interfering RNA (LVi-Cdk5) exerted neuroprotection by inhibiting abnormal mitochondrial fission through Drp1 phosphorylation at Ser616 and mitochondrial translocation after chronic ethanol exposure. Taken together, the present study demonstrated that inhibition of aberrant Cdk5 activation attenuates hippocampal neuron injury and cognitive deficits induced by chronic exposure to ethanol through Drp1-mediated mitochondrial fission and mitochondrial dysfunction. Interfering with this pathway might serve as a potential therapeutic approach to prevent ethanol-induced neurotoxicity in the brain.

CDK5 Mediates Proinflammatory Effects of Microglia through Activated DRP1 Phosphorylation in Rat Model of Intracerebral Hemorrhage.

Introduction Cyclin-dependent kinase-5 (CDK5) is a key kinase involved in brain development and function and recently found to be involved in neuronal and astroglial apoptosis, neural stem/progenitor cell stemness, mitochondrial fission, and synaptic transmission. But the specific mechanism of CDK5-mediated anti-inflammatory remains unclear in ICH. The aim of the present study was to explore the role of CDK5 in mediating microglia activity through activated DRP1 phosphorylation in a rat ICH model. Methods We measured behavioral change after ICH; detected the expression of CDK5 in the rat brain using immunohistochemistry; and measured the protein levels of CDK5, p35, p25, p-histone H1, and p-DRP1 using Western blot analysis. Coimmunoprecipitation analysis indicated interaction of CDK5 and DRP1. Tumor necrosis factor-α, interleukin- (IL-) 1β, and IL-6 levels were measured using enzyme-linked immunosorbent assay (ELISA). Results After ICH, CDK5 protein level and kinase activity increased. Western blot data showed that CDK5 expression increased from 6 h and peaked at 2 d after ICH (p < 0.05), and the expression of p35 was lowest at 12 h, while the expression of p25 peaked at 2 d after ICH. Besides, p-DRP1 expression change follows with CDK5 kinase activity change. Coimmunoprecipitation showed that interaction between CDK5 and DRP1 certainly exists in microglia. Then, knockdown CDK5 or p35 expression by siRNA reduced the expression level of p-DRP1. ELISA data showed that the protein levels of proinflammatory mediators, such as TNF-α, IL-1β, and IL-6, were decreased by knockdown of CDK5. Conclusion CDK5 may regulate DRP1 by direct phosphorylation in microglia and further induce microglia secreting proinflammation factor.

Abstract 99: Interplay between CDK5 and IRF2BP1 enhances breast cancer brain metastasis

Abstract Prognosis of patients with breast cancer brain metastasis is poor due to unavailability of effective treatments. Hence, more fundamental research is needed to broaden our understanding of mechanisms underlying the origin and development of brain metastasis. We have identified that expression of cyclin-dependent kinase 5 (CDK5) in cancer cells promotes brain metastasis growth in experimental mouse models through suppressing the antitumor immunity. Here, we interrogated the underlying mechanism that connects CDK5 with defective antitumor immunity. To understand the repertoire of CDK5 phosphorylation targets, we performed a quantitative stable isotope labeling by amino acids in cell culture (SILAC) phopshoproteomics analysis of cultured brain-seeking cell lines that have been genetically engineered to express shRNA targeting the CDK5 gene. The knockdown of CDK5 resulted in a dramatic dephosphorylation of multiple proteins, among which Interferon Regulatory Factor 2 Binding Protein 1 (Irf2bp1) is well recognized to function in mediating the immune response. Knockdown of CDK5 reduced the phosphorylation of Irf2bp1 at Ser66 in both 4T1 and T11 cells (Log10 fold change: -0.51 and -0.39, respectively, P less than 0.0001). These findings were further validated by co-immunoprecipitation of Irf2bp1 in cell lysates from shCtl and shCDK5 brain-seeking cancer cell lines, followed by immunoblotting for phosphoserine. Furthermore, analysis of Irf2bp1 protein band shifts and intensities using Phos-tag™ gels also demonstrated that CDK5 knockdown reduces the Irf2bp1 phosphorylation. In order to investigate the function of Irf2bp1, we established a stable knockdown of the Irf2bp1 gene in brain metastasis-derived cell lines 4T1.Br3 and T11.Br1. We observed that Irf2bp1-knockdown cells displayed a reduced expression of genes encoding the class I major histocompatibility complex (MHC-I) as well as Nlrc5, a key transcriptional regulator of MHC I. Thus, these findings indicate that CDK5 may regulate the expression of surface MHC-I in breast cancer brain metastasis cells through Irf2bp1 phosphorylation at Ser66, in the absence of Interferon gamma or other classically recognized MHC-I agonists. These findings reveal a previously unknown mechanism of MHC-I regulation in cancer cells, that might improve our knowledge about cancer cell immune evasion through MHC-I loss. Citation Format: Arseniy E. Yuzhalin, Frank Lowery, Yohei Saito, Patrick Zhang, Dihua Yu. Interplay between CDK5 and IRF2BP1 enhances breast cancer brain metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 99.

Downregulation of CDK5 signaling in the dorsal striatum alters striatal microcircuits implicating the association of pathologies with circadian behavior in mice

Dysfunction of striatal dopaminergic circuits has been implicated in motor impairment and Parkinson’s disease (PD)-related circadian perturbations that may represent an early prodromal marker of PD. Cyclin-dependent kinase 5 (CDK5) negatively regulates dopamine signaling in the striatum, suggesting a critical role of CDK5 in circadian and sleep disorders. Here, we used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing to produce mice with a dorsal striatum (DS)-specific knockdown (KD) of the Cdk5 gene (referred to as DS-CDK5-KD mice) and investigate its role in vivo. DS-CDK5-KD mice exhibited deficits in locomotor activity and disturbances in activity/rest behavior. Additionally, Golgi staining of neurons in the DS revealed that CDK5 deletion reduced dendrite length and the number of functional synapses, which was confirmed by significant downregulation of MAP2, PSD-95, and synapsin I. Correlated with this, DS-CDK5-KD mice displayed reduced phosphorylation of Tau at Thr181. Furthermore, whole-cell patch-clamp recordings of green fluorescent protein-tagged neurons in the striatum of DS-CDK5-KD mice revealed a decreased frequency of spontaneous inhibitory postsynaptic currents and altered excitatory/inhibitory synaptic balance. Notably, anterograde labeling showed that CDK5 KD in the DS disrupted long-range projections to the secondary motor cortex, dorsal and ventral thalamic nuclei, and basolateral amygdala, which are involved in the regulation of motor and circadian rhythms in the brain. These findings support a critical role of CDK5 in the DS in maintaining the striatal neural circuitry underlying motor functions and activity/rest associated with circadian rhythms that are perturbed in neurodegenerative disorders.

Inhibition of Cdk5 increases osteoblast differentiation and bone mass and improves fracture healing

Identification of regulators of osteoblastogenesis that can be pharmacologically targeted is a major goal in combating osteoporosis, a common disease of the elderly population. Here, unbiased kinome RNAi screening in primary murine osteoblasts identified cyclin-dependent kinase 5 (Cdk5) as a suppressor of osteoblast differentiation in both murine and human preosteoblastic cells. Cdk5 knockdown by siRNA, genetic deletion using the Cre-loxP system, or inhibition with the small molecule roscovitine enhanced osteoblastogenesis in vitro. Roscovitine treatment significantly enhanced bone mass by increasing osteoblastogenesis and improved fracture healing in mice. Mechanistically, downregulation of Cdk5 expression increased Erk phosphorylation, resulting in enhanced osteoblast-specific gene expression. Notably, simultaneous Cdk5 and Erk depletion abrogated the osteoblastogenesis conferred by Cdk5 depletion alone, suggesting that Cdk5 regulates osteoblast differentiation through MAPK pathway modulation. We conclude that Cdk5 is a potential therapeutic target to treat osteoporosis and improve fracture healing.

Cyclin-dependent kinase 5 promotes the growth of tongue squamous cell carcinoma through the microRNA 513c-5p/cell division cycle 25B pathway and is associated with a poor prognosis.

BackgroundThe objective of this study was to investigate the role and molecular mechanism of cyclin‐dependent kinase 5 (CDK5) in regulating the growth of tongue squamous cell carcinoma (TSCC).MethodsThe authors used multiple methods to detect the levels of CDK5 expression in samples of TSCC and to explore the relation between CDK5 expression and various clinicopathologic factors. In vivo and in vitro cell experiments were performed to detect the proliferation, invasion, and migration of TSCC cells with CDK5 knockdown or overexpression. These studies verified that CDK5 regulates the occurrence and development of TSCC cells through the microRNA 513c‐5p/cell division cycle 25B pathway.ResultsAn elevated level of CDK5 expression in TSCC tissues was identified as an independent risk factor affecting TSCC growth and patient prognosis. Patients who had TSCC with low levels of CDK5 expression had a higher survival rate than those with high levels. Knockdown of CDK5 reduced the proliferation, migration, and invasion of TSCC cells both in vitro and in vivo. In addition, the authors observed that CDK5 regulated the growth of TSCC through the microRNA 513c‐5p/cell division cycle C25B pathway.ConclusionsCDK5 functions as an oncogene in TSCC and might serve as a molecular marker for use in the diagnosis and treatment of TSCC.Lay Summary Tongue squamous cell carcinoma (TSCC) is 1 of the most common malignant tumors of the head and neck, and the survival rate of patients with tongue cancer has been very low.Therefore, it is important to study the molecular mechanism of TSCC progression to identify biomarkers that can be used to improve its clinical diagnosis and treatment.Cyclin‐dependent kinase 5 (CDK5) is an atypical member of the cyclin‐dependent kinase family and is involved in regulating the cell cycle.Changes in the cell cycle are of great significance for the occurrence and development of tumor cells; and, in recent years, increasing evidence has suggested that CDK5 exists in a disordered state in cancer cells.In this study, the authors demonstrate that CDK5 functions as an oncogene in TSCC and might serve as a molecular marker for use in the diagnosis and treatment of TSCC.

Inhibition of Cdk5 Ameliorates Skeletal Bone Loss in Glucocorticoid-Treated Mice.

Glucocorticoids (GCs) are widely used to treat inflammatory diseases. However, their long-term use leads to glucocorticoid-induced osteoporosis, increasing morbidity and mortality. Both anabolic and anti-resorptive drugs are used to counteract GC-induced bone loss, however, they are expensive and/or have major side effects. Therefore, identifying new targets for cost-effective, small-molecule inhibitors is essential. We recently identified cyclin-dependent kinase 5 (Cdk5) as a suppressor of osteoblast differentiation and showed that its inhibition with roscovitine promoted osteoblastogenesis, thus improving the skeletal bone mass and fracture healing. Here, we assessed whether Cdk5 knockdown or inhibition could also reverse the GC-mediated suppression of osteoblast differentiation, bone loss, and fracture healing. We first demonstrated that Cdk5 silencing abolished the dexamethasone (Dex)-induced downregulation of alkaline phosphatase (Alp) activity, osteoblast-specific marker gene expression (Runx2, Sp7, Alpl, and Bglap), and mineralization. Similarly, Cdk5 inhibition rescued Dex-induced suppression of Alp activity. We further demonstrated that Cdk5 inhibition reversed prednisolone (Pred)-induced bone loss in mice, due to reduced osteoclastogenesis rather than improved osteoblastogenesis. Moreover, we revealed that Cdk5 inhibition failed to improve Pred-mediated impaired fracture healing. Taken together, we demonstrated that Cdk5 inhibition with roscovitine ameliorated GC-mediated bone loss but did not reverse GC-induced compromised fracture healing in mice.

Inhibition of Cdk5 in PV Neurons Reactivates Experience-Dependent Plasticity in Adult Visual Cortex.

Cyclin-dependent kinase 5 (Cdk5) has been shown to play a critical role in brain development, learning, memory and neural processing in general. Cdk5 is widely distributed in many neuron types in the central nervous system, while its cell-specific role is largely unknown. Our previous study showed that Cdk5 inhibition restored ocular dominance (OD) plasticity in adulthood. In this study, we specifically knocked down Cdk5 in different types of neurons in the visual cortex and examined OD plasticity by optical imaging of intrinsic signals. Downregulation of Cdk5 in parvalbumin-expressing (PV) inhibitory neurons, but not other neurons, reactivated adult mouse visual cortical plasticity. Cdk5 knockdown in PV neurons reduced the evoked firing rate, which was accompanied by an increment in the threshold current for the generation of a single action potential (AP) and hyperpolarization of the resting membrane potential. Moreover, chemogenetic activation of PV neurons in the visual cortex can attenuate the restoration of OD plasticity by Cdk5 inhibition. Taken together, our results suggest that Cdk5 in PV interneurons may play a role in modulating the excitation and inhibition balance to control the plasticity of the visual cortex.

Targeting CDK5 in Astrocytes Promotes Calcium Homeostasis Under Excitotoxic Conditions.

Glutamate excitotoxicity triggers overactivation of CDK5 and increases calcium influx in neural cells, which promotes dendritic retraction, spine loss, increased mitochondrial calcium from the endoplasmic reticulum, and neuronal death. Our previous studies showed that CDK5 knockdown (KD) in astrocytes improves neurovascular integrity and cognitive functions and exerts neuroprotective effects. However, how CDK5-targeted astrocytes affect calcium regulation and whether this phenomenon is associated with changes in neuronal plasticity have not yet been analyzed. In this study, CDK5 KD astrocytes transplanted in CA3 remained at the injection site without proliferation, regulated calcium in the CA1 hippocampal region after excitotoxicity by glutamate in ex vivo hippocampal slices, improving synapsin and PSD95 clustering. These CDK5 KD astrocytes induced astrocyte stellation and neuroprotection after excitotoxicity induced by glutamate in vitro. Also, these effects were supported by CDK5 inhibition (CDK5i) in vitro through intracellular stabilization of calcium levels in astrocytes. Additionally, these cells in cocultures restored calcium homeostasis in neurons, redistributing calcium from somas to dendrites, accompanied by dendrite branching, higher dendritic spines and synapsin-PSD95 clustering. In summary, induction of calcium homeostasis at the CA1 hippocampal area by CDK5 KD astrocytes transplanted in the CA3 area highlights the role of astrocytes as a cell therapy target due to CDK5-KD astrocyte-mediated synaptic clustering, calcium spreading regulation between both areas, and recovery of the intracellular astrocyte-neuron calcium imbalance and plasticity impairment generated by glutamate excitotoxicity.

Knockdown of CDK5 down-regulates PD-L1 via the ubiquitination-proteasome pathway and improves antitumor immunity in lung adenocarcinoma

Although immunotherapy (anti-PD-1/PD-L1 antibodies) has been approved for clinical treatment of lung cancer, only a small proportion of patients respond to monotherapy. Hence, understanding the regulatory mechanism of PD-L1 is particularly important to identify optimal combinations. In this study, we found that inhibition of CDK5 induced by shRNA or CDK5 inhibitor leads to reduced expression of PD-L1 protein in human lung adenocarcinoma cells, while the mRNA level is not substantially altered. The PD-L1 protein degradation is mediated by E3 ligase TRIM21 via ubiquitination-proteasome pathway. Subsequently, we studied the function of CDK5/PD-L1 axis in LUAD. In vitro, the absence of CDK5 in mouse Lewis lung cancer cell (LLC) has no effect on cell proliferation. However, the attenuation of CDK5 or combined with anti-PD-L1 greatly suppresses tumor growth in LLC implanted mouse models in vivo. Disruption of CDK5 elicits a higher level of CD3+, CD4+ and CD8+ T cells in spleens and lower PD-1 expression in CD4+ and CD8+ T cells. Our findings highlight a role for CDK5 in promoting antitumor immunity, which provide a potential therapeutic target for combined immunotherapy in LUAD.

Lipopolysaccharide-induced inflammation in human peritoneal mesothelial cells is controlled by ERK1/2-CDK5-PPARγ axis.

BackgroundPeritonitis is a common complication in which the peritoneum becomes inflamed. Peroxisome proliferator-activated receptor (PPAR)γ agonists and extracellular signal-regulated kinases 1/2 (ERK1/2) inactivation have been found to restore damage caused by lipopolysaccharide-induced (LPS) inflammation. This study aimed to investigate the association between PPARγ and ERK1/2 in LPS-induced inflammation in peritonitis.MethodsHuman peritoneal mesothelial cells were maintained in Dulbecco’s Modified Eagle Medium and treated with LPS under a series of different concentrations and treatment times. Cellular interleukins-1βeta (IL-1β), cellular interleukins-6 (IL-6), cellular interleukins-12 (IL-12) were measured by enzyme-linked immunosorbent assay (ELISA) assay. Expression or activation of cyclin-dependent kinase (CDK)5, ERK1/2, and PPARγ was detected using quantitative real-time PCR and/or western blot.ResultsLPS induced dose- and time-dependent increments in the cellular IL-1β, IL-6, and IL-12 contents, cyclin-dependent kinase 5 (CDK5) expression, and PPARγSer273 phosphorylation. Treatment with 1 µg/mL LPS for 12 hours was the optimal experimental design for inflammation stimulation. The concentration of LPS over 1 µg/mL or treatment more than 12 hours reduced the inflammatory status. LPS stimulation also activated ERK1/2 and increased its interaction with CDK5. Further, ERK1/2 inhibition by AZD0364 prevented IL-1β, IL-6, IL-12, and CDK5 expression, as well as activation of ERK1/2 and phosphorylation of PPARγ, induced by LPS. Knockdown of CDK5 using its siRNA caused similar changes as AZD0364, minus ERK1/2 inactivation.ConclusionsOur results suggested that LPS-induced inflammation in human peritoneal mesothelial cells can be partly suppressed by inhibiting the ERK1/2/CDK5/PPARγ axis.

Fusobacterium nucleatum Promotes the Progression of Colorectal Cancer Through Cdk5-Activated Wnt/β-Catenin Signaling.

Background/AimsGrowing evidence supports the direct link of Fusobacterium nucleatum with colorectal cancer (CRC). However, to date, the underlying mechanism of action remains poorly understood. In this study, we examined the effects of F. nucleatum on the progression of CRC and investigated whether cyclin-dependent kinase 5 (Cdk5) is involved in the effect through activating the Wnt/β-catenin signaling pathway.Materials and MethodsCRC tissues and matched histologically normal specimens were collected from patients who were diagnosed with CRC and underwent surgical treatment in our hospital between January 2018 and January 2019. Two human CRC cell lines, including DLD-1 and SW480, were utilized mainly for in vitro mechanistic investigations.ResultsThe abundance of F. nucleatum was significantly greater in CRC tissues than in cancer-free specimens, which was significantly correlated with the progression of CRC. In vitro investigations revealed that F. nucleatum significantly enhanced the proliferation and migration of CRC cells. Furthermore, F. nucleatum significantly induced the expression of Cdk5 and activation of the Wnt/β-catenin signaling pathway. Notably, knockdown of Cdk5 significantly abrogated the effects of F. nucleatum on cellular processes and Wnt/β-catenin signaling in relation to the progression of CRC.ConclusionThe results of this study demonstrate that F. nucleatum orchestrates a molecular network involving the direct role of Cdk5 in activating Wnt/β-catenin signaling to modulate CRC progression. Thus, in-depth investigations of F. nucleatum-associated molecular pathways may offer valuable insight into the pathogenesis of CRC, which may help further the development of treatment for this disease.

CDK5 Knockdown inhibits proliferation and induces apoptosis and Cell Cycle Arrest in Human Glioblastoma.

Aims: Gliomas are the most common malignant brain neoplasms with high recurrence and lethality rates. Recently, studies have reported that cyclin-dependent kinase 5 (CDK5) is involved in tumorigenesis. Herein, we applied bioinformatics analysis to determine the clinical value of CDK5 in patients with glioma and examined the effects of CDK5 on glioblastoma cell proliferation, apoptosis, and cell cycle in vitro.Methods: Gene expression profiles containing clinical data of low-grade glioma (LGG) and glioblastoma cohorts were obtained from The Cancer Genome Atlas database and analyzed to determine the association between CDK5 expression and glioma clinicopathological characteristics. Kaplan-Meier survival analysis was performed for prognosis analysis. Gene set enrichment analysis (GSEA) was used to identify the biological pathways involved in differential CDK5 expression. In vitro experiments were performed to explore the effects of CDK5 on glioma cell functions.Results: CDK5 expression was substantially higher in glioblastoma than in LGG. GSEA showed that some metabolism-related pathways were associated with the high CDK5 expression phenotype. In vitro experiments showed that CDK5 knockdown impaired cell proliferation and colony formation ability, and induced apoptosis and cell cycle arrest.Conclusion: CDK5 may act as a potential biomarker of glioma progression and a valid target for glioma therapy.

Cyclin-dependent Kinase 5 Regulates Cortical Neurotransmission and Neural Circuits Associated with Motor Control in the Secondary Motor Cortex in the Mouse

Cyclin-dependent kinase 5 (Cdk5) is a regulator of axon growth and radial neuronal migration in the developing mouse brain, and it plays critical roles in cortical structure formation and brain function. However, the function of Cdk5 in cortico-cortical and cortico-sensorimotor networks in the adult remains largely unknown. In this study, we investigated the function of Cdk5 in the rostral secondary motor cortex (M2) in the male mouse using CRISPR/Cas9 gene editing and somatic brain transgenesis, to produce M2-specific knockdown of Cdk5 in neurons in the male mouse. Mouse deficient in Cdk5 in the M2 exhibited a reduction in both the number of functional synapses and the total basal dendritic length, as well as motor dysfunction. Furthermore, whole-cell patch-clamp recordings in layer V green fluorescent protein (GFP)-tag pyramidal neurons revealed a decrease in the frequency and amplitude of miniature EPSCs and miniature IPSCs, as well as a reduction in the population synaptic responses (fEPSPs) in these mice. Specifically, retrograde labeling showed that Cdk5 knockdown in the M2 caused a reduction in long-range projections to the M2 from the thalamus/prefrontal cortex and claustrum. Collectively, our findings show a new regulatory role of Cdk5 in neural circuit maintenance, and that the changes in neural transmission and circuits in the mice with Cdk5 knockdown in the M2 likely contribute to the motor dysfunction in these animals.

Cyclin-dependent kinase 5 (CDK5) regulates the circadian clock.

Circadian oscillations emerge from transcriptional and post-translational feedback loops. An important step in generating rhythmicity is the translocation of clock components into the nucleus, which is regulated in many cases by kinases. In mammals, the kinase promoting the nuclear import of the key clock component Period 2 (PER2) is unknown. Here, we show that the cyclin-dependent kinase 5 (CDK5) regulates the mammalian circadian clock involving phosphorylation of PER2. Knock-down of Cdk5 in the suprachiasmatic nuclei (SCN), the main coordinator site of the mammalian circadian system, shortened the free-running period in mice. CDK5 phosphorylated PER2 at serine residue 394 (S394) in a diurnal fashion. This phosphorylation facilitated interaction with Cryptochrome 1 (CRY1) and nuclear entry of the PER2-CRY1 complex. Taken together, we found that CDK5 drives nuclear entry of PER2, which is critical for establishing an adequate circadian period of the molecular circadian cycle. Of note is that CDK5 may not exclusively phosphorylate PER2, but in addition may regulate other proteins that are involved in the clock mechanism. Taken together, it appears that CDK5 is critically involved in the regulation of the circadian clock and may represent a link to various diseases affected by a derailed circadian clock.

CDK5 neutralizes the tumor suppressing effect of BIN1 via mediating phosphorylation of c-MYC at Ser-62 site in NSCLC

BackgroundBridging integrator 1 (BIN1) has showed outstanding tumor-suppressive potential via inhibiting c-MYC-mediated tumorigenesis. However, a frequent phosphorylation of c-MYC at Ser-62 site could block the BIN1/c-MYC interaction and limits the tumor-suppressive effect of BIN1. Cyclin-dependent kinase 5 (CDK5), a generally dysregulated protein in various carcinomas, can mediate c-MYC phosphorylation at Ser-62 site. However, whether the existence of CDK5 could block the BIN1/c-MYC interaction remains unclear.Materials and methodsThe expression of CDK5 and BIN1 in non-small cell lung cancer (NSCLC) cell lines were measured. CDK5 was knocked down and overexpressed in H460 and PC9 cells, respectively. CCK-8, wound healing and transwell were used to detect the proliferation, migration and invasion ability of NSCLC cells. Tumor-bearing nude mouse model was built with H460 cells. Dinaciclib was added to realize the effect of CDK5 inhibition in vivo. NSCLC and matched para-carcinoma specimens were collected from 153 patients who underwent radical operation. IHC was performed to determine the expression of CDK5 in the specimens. Kaplan–Meier analysis was used to analyze the correlation between the postoperative survival and CDK5 expression.ResultsCDK5 was highly expressed in H460 cells, and knockdown of CDK5 could restore the BIN1/c-MYC interaction. Meanwhile, low expression of CDK5 was observed in PC9 cells, and overexpression of CDK5 blocked the BIN1/c-MYC interaction. Consequently, the growth, migration, invasion and epithelial mesenchymal transition (EMT) ability of H460 and PC9 cells could be facilitated by CDK5. The addition of CDK5 inhibitor Dinaciclib significantly suppressed the tumorigenesis ability of NSCLC cells in tumor-bearing mouse model. Furthermore, high expression of CDK5, along with low expression of BIN1, could predict poor postoperative prognosis of NSCLC patients. The patients with high expression of CDK5 and low expression of BIN1 showed similar prognosis, indicating that CDK5 could neutralize the tumor suppressing effect of BIN1 in clinical situation.ConclusionsCDK5 blocked the interaction of BIN1 and c-MYC via promoting phosphorylation of c-MYC at ser-62 site, ultimately facilitated the progression of NSCLC.

La pérdida de función de la quinasa dependiente de ciclina 5 (CDK5) altera el citoesqueleto y reduce la infección in vitro por el virus del dengue 2

La quinasa dependiente de ciclina 5 (CDK5) regula diversas funciones en neuronas, células endoteliales y epiteliales, entre ellas la dinámica del citoesqueleto. Así mismo, se ha reportado que componentes del citoesqueleto, tales como, filamentos de actina y microtúbulos juegan un rol importante durante la infección por el virus dengue (DENV). El objetivo del presente trabajo fue evaluar por dos métodos, inhibición química y silenciamiento génico, la participación de CDK5 durante la infección por DENV-2. La actividad antiviral de roscovitina fue evaluada usando ensayos de Unidades Formadoras de Placa (PFU). La eficiencia de transfección y el silenciamiento de CDK5, empleando miARNs artificiales, se determinó por citometría de flujo. El efecto sobre la proteína de envoltura viral y elementos del citoesqueleto se evidenció mediante microscopia avanzada de fluorescencia y análisis de imágenes. Roscovitina mostró actividad antiviral en etapas pre y post-infectivas en una forma dependiente de la dosis. El tratamiento con roscovitina y miRCDK5 mostró ser efectivo reduciendo la cantidad de CDK5 en células no infectadas. En células infectadas y transfectadas con miRCDK5, así como tratadas con el inhibidor, se observó una reducción significativa de la proteína de envoltura viral; sin embargo, no se encontró reducción significativa de CDK5. Además, el tratamiento con roscovitina indujo cambios celulares morfológicos evidentes en células infectadas. Los resultados indican la potencial participación de CDK5 durante la infección por DENV-2, posiblemente mediando la traducción proteica o la replicación del genoma viral a través de la regulación de la dinámica del citoesqueleto. Se requieren datos adicionales para esclarecer la mecanística del fenómeno usando métodos alternativos.

CDK5 Inhibitor Downregulates Mcl-1 and Sensitizes Pancreatic Cancer Cell Lines to Navitoclax.

Developing small molecules that indirectly regulate Mcl-1 function has attracted a lot of attention in recent years. Here, we report the discovery of an aminopyrazole, 2-([1,1′-biphenyl]-4-yl)-N-(5-cyclobutyl-1H-pyrazol-3-yl)acetamide (analog 24), which selectively inhibited cyclin-dependent kinase (CDK) 5 over CDK2 in cancer cell lines. We also show that analog 24 reduced Mcl-1 levels in a concentration-dependent manner in cancer cell lines. Using a panel of doxycycline inducible cell lines, we show that CDK5 inhibitor 24 selectively modulates Mcl-1 function while the CDK4/6 inhibitor 6-acetyl-8-cyclopentyl-5-methyl-2-(5-(piperazin-1-yl)pyridin-2-ylamino)pyrido[2,3-day]pyrimidin-7(8H)-one does not. Previous studies using RNA interference and CRISPR showed that concurrent elimination of Bcl-xL and Mcl-1 resulted in induction of apoptosis. In pancreatic cancer cell lines, we show that either CDK5 knockdown or expression of a dominant negative CDK5 when combined with Bcl2 inhibitor results in synergistic induction of apoptosis. Moreover, concurrent pharmacological perturbation of Mcl-1 and Bcl-xL in pancreatic cancer cell lines using a CDK5 inhibitor analog 24 that reduced Mcl-1 levels and 4-(4-{[2-(4-chlorophenyl)-5,5-dimethyl-1-cyclohexen-1-yl]methyl}-1-piperazinyl)-N-[(4-{[(2R)-4-(4-morpholinyl)-1-(phenylsulfanyl)-2-butanyl]amino}-3-[(trifluoromethyl)sulfonyl]phenyl)sulfonyl] benzamide (navitoclax), a Bcl-2/Bcl-xL/Bcl-w inhibitor, resulted in synergistic inhibition of cell growth and induction of apoptosis. In conclusion, we demonstrate targeting CDK5 will sensitize pancreatic cancers to Bcl-2 inhibitors.SIGNIFICANCE STATEMENTMcl-1 is stabilized by CDK5-mediated phosphorylation in pancreatic ductal adenocarcinoma, resulting in the deregulation of the apoptotic pathway. Thus, genetic or pharmacological targeting of CDK5 sensitizes pancreatic cancers to Bcl-2 inhibitors, such as navitoclax.