Biological functions of CDK5 and potential CDK5 targeted clinical treatments.

Cyclin dependent kinases are proline-directed serine/threonine protein kinases that are traditionally activated upon association with a regulatory subunit. For most CDKs, activation by a cyclin occurs through association and phosphorylation of the CDK\u27s T-loop. CDK5 is unusual because it is not typically activated upon binding with a cyclin and does not require T-loop phosphorylation for activation, even though it has high amino acid sequence homology with other CDKs. While it was previously thought that CDK5 only interacted with p35 or p39 and their cleaved counterparts, Recent evidence suggests that CDK5 can interact with certain cylins, amongst other proteins, which modulate CDK5 activity levels. This review discusses recent findings of molecular interactions that regulate CDK5 activity and CDK5 associated pathways that are implicated in various diseases. Also covered herein is the growing body of evidence for CDK5 in contributing to the onset and progression of tumorigenesis

The CCL5/CCR5 axis promotes metastasis in basal breast cancer.

Recently, we have shown that the CCL5/CCR5 axis is active in patients affected by an aggressive basal subtype of breast cancer. Using preclinical models, we have demonstrated that CCR5 promotes breast cancer invasiveness and metastatic potential, while CCR5 inhibition abrogates them. Thus, CCR5 antagonists may constitute an alternative therapeutic approach for patients affected by metastatic basal breast cancer

Inducible transgenics. New lessons on events governing the induction and commitment in mammary tumorigenesis

Breast cancer arises from multiple genetic events that together contribute to the established, irreversible malignant phenotype. The development of inducible tissue-specific transgenics has allowed a careful dissection of the events required for induction and subsequent maintenance of tumorigenesis. Mammary gland targeted expression of oncogenic Ras or c-Myc is sufficient for the induction of mammary gland tumorigenesis in the rodent, and when overexpressed together the rate of tumor onset is substantially enhanced. In an exciting recent finding, D'Cruz et al discovered tetracycline-regulated c-Myc overexpression in the mammary gland induced invasive mammary tumors that regressed upon withdrawal of c-Myc expression. Almost one-half of the c-Myc-induced tumors harbored K-ras or N-ras gene point mutations, correlating with tumor persistence on withdrawal of c-Myc transgene expression. These findings suggest maintenance of tumorigenesis may involve a second mutation within the Ras pathway

SIRT1 modulates aggregation and toxicity through deacetylation of the androgen receptor in cell models of SBMA

Posttranslational protein modifications can play a major role in disease pathogenesis; phosphorylation, sumoylation, and acetylation modulate the toxicity of a variety of proteotoxic proteins. The androgen receptor (AR) is substantially modified, in response to hormone binding, by phosphorylation, sumoylation, and acetylation; these modifications might thus contribute to DHT-dependent polyglutamine (polyQ)-expanded AR proteotoxicity in spinal and bulbar muscular atrophy (SBMA). SIRT1, a nuclear protein and deacetylase of the AR, is neuroprotective in many neurodegenerative disease models. Our studies reveal that SIRT1 also offers protection against polyQexpanded AR by deacetylating the AR at lysines 630/632/633. This finding suggested that nuclear AR acetylation plays a role in the aberrant metabolism and toxicity of polyQ-expanded AR. Subsequent studies revealed that the polyQ-expanded AR is hyperacetylated and that pharmacologic reduction of acetylation reduces mutantARaggregation. Moreover, genetic mutation to inhibit polyQ-expanded ARacetylation of lysines 630/632/633 substantially decreased its aggregation and completely abrogated its toxicity in cell lines and motor neurons. Our studies also reveal one means by which the AR acetylation state likely modifies polyQ-expanded AR metabolism and toxicity, through its effect on DHT-dependent AR stabilization. Overall, our findings reveal a neuroprotective function of SIRT1 that operates through its deacetylation of polyQ-expanded AR and highlight the potential of both SIRT1 and AR acetylation as powerful therapeutic targets in SBMA. © 2011 the authors

Cyclin D1-mediated microRNA expression signature predicts breast cancer outcome

Background: Genetic classification of breast cancer based on the coding mRNA suggests the evolution of distinct subtypes. Whether the non-coding genome is altered concordantly with the coding genome and the mechanism by which the cell cycle directly controls the non-coding genome is poorly understood. Methods: Herein, the miRNA signature maintained by endogenous cyclin D1 in human breast cancer cells was defined. In order to determine the clinical significance of the cyclin D1-mediated miRNA signature, we defined a miRNA expression superset from 459 breast cancer samples. We compared the coding and non-coding genome of breast cancer subtypes. Results: Hierarchical clustering of human breast cancers defined four distinct miRNA clusters (G1-G4) associated with distinguishable relapse-free survival by Kaplan-Meier analysis. The cyclin D1-regulated miRNA signature included several oncomirs, was conserved in multiple breast cancer cell lines, was associated with the G2 tumor miRNA cluster, ERα+ status, better outcome and activation of the Wnt pathway. The coding and non-coding genome were discordant within breast cancer subtypes. Seed elements for cyclin D1-regulated miRNA were identified in 63 genes of the Wnt signaling pathway including DKK. Cyclin D1 restrained DKK1 via the 3\u27UTR. In vivo studies using inducible transgenics confirmed cyclin D1 induces Wnt-dependent gene expression. Conclusion: The non-coding genome defines breast cancer subtypes that are discordant with their coding genome subtype suggesting distinct evolutionary drivers within the tumors. Cyclin D1 orchestrates expression of a miRNA signature that induces Wnt/β-catenin signaling, therefore cyclin D1 serves both upstream and downstream of Wnt/β-catenin signaling

Alternate cyclin D1 mRNA splicing modulates P27\u3csup\u3eKlP1\u3c/sup\u3e binding and cell migration

Cyclin D1 is an important cell cycle regulator but in cancer its overexpression also increases cellular migration mediated by p27KlP1 stabilization and RhoA inhibition. Recently, a common polymorphism at the exon 4-intron 4 boundary of the human cyclin D1 gene within a splice donor region was associated with an altered risk of developing cancer. Altered RNA splicing caused by this polymorphism gives rise to a variant cyclin D1 isoform termed cyclin D1b, which has the same N-terminus as the canonical cyclin D1a isoform but a distinct C-terminus. In this study we show that these different isoforms have unique properties with regard to the cellular migration function of cyclin D1. Whereas they displayed little difference in transcriptional co-repression assays on idealized reporter genes, microarray cDNA expression analysis revealed differential regulation of genes including those that influence cellular migration. Additionally, while cyclin D1a stabilized p27KIP1 and inhibited RhoA-induced ROCK kinase activity, promoting cellular migration, cyclin D1b failed to stabilize p27KIP1 or inhibit ROCK kinase activity and had no effect on migration. Our findings argue that alternate splicing is an important determinant of the function of cyclin D1 in cellular migration

Dual fluorescent molecular substrates selectively report the activation, sustainability and reversibility of cellular PKB/Akt activity

Using a newly developed near-infrared (NIR) dye that fluoresces at two different wavelengths (dichromic fluorescence, DCF), we discovered a new fluorescent substrate for Akt, also known as protein kinase B, and a method to quantitatively report this enzyme\u27s activity in real time. Upon insulin activation of cellular Akt, the enzyme multi-phosphorylated a single serine residue of a diserine DCF substrate in a time-dependent manner, culminating in monophospho- to triphospho-serine products. The NIR DCF probe was highly selective for the Akt1 isoform, which was demonstrated using Akt1 knockout cells derived from MMTV-ErbB2 transgenic mice. The DCF mechanism provides unparalleled potential to assess the stimulation, sustainability, and reversibility of Akt activation longitudinally. Importantly, NIR fluorescence provides a pathway to translate findings from cells to living organisms, a condition that could eventually facilitate the use of these probes in humans

Ras Inactivation of the Retinoblastoma Pathway by Distinct Mechanisms in NIH 3T3 Fibroblast and RIE-1 Epithelial Cells

Although Ras and Raf cause transformation of NIH 3T3 fibroblasts, only Ras causes transformation of RIE-1 intestinal epithelial cells. To determine if the inability of Raf to transform RIE-1 cells is due to a failure to deregulate cell cycle progression, we evaluated the consequences of sustained Ras and Raf activation on steady state levels of cyclin D1, p21(CIP/WAF), and p27(KIP1). Both Ras- and Raf-transformed NIH 3T3 cells showed up-regulated expression of cyclin D1, p21, and p27 protein, increased retinoblastoma (Rb) hyperphosphorylation, and increased activation of E2F-mediated transcription. Similarly, Ras-transformed RIE-1 cells also showed up-regulation of cyclin D1, p21, and hyperphosphorylated Rb. In contrast, Ras-mediated down-regulation of p27 was seen in RIE-1 cells. Conversely, stable expression of activated Raf alone caused only a partial up-regulation of p21 and Rb hyperphosphorylation but no activation of E2F-responsive transcription or down-regulation of p27 in RIE-1 cells. Moreover, we found that the AP-1 site was dispensable for Ras-mediated stimulation of the cyclin-D1 promoter in NIH 3T3 cells but was essential for Ras-mediated stimulation in RIE-1 cells. Thus, up-regulation of p21, rather than the down-regulation seen in previous transient expression studies, is seen with sustained Ras activation. Additionally, p27 may serve a positive (NIH 3T3) or negative (RIE-1) regulatory role in Ras transformation that is cell type-dependent. The involvement of Raf and phosphatidylinositol 3-kinase in mediating Ras changes in cyclin D1, p21, and p27 was also very distinct in NIH 3T3 and RIE-1 cells. Taken together, these results demonstrate the importance of Raf-independent pathways in mediating oncogenic Ras deregulation of cell cycle progression in epithelial cells

Cyclin D1 Restrains Oncogene-Induced Autophagy by Regulating the AMPK-LKB1 Signaling Axis.

Autophagy activated after DNA damage or other stresses mitigates cellular damage by removing damaged proteins, lipids, and organelles. Activation of the master metabolic kinase AMPK enhances autophagy. Here we report that cyclin D1 restrains autophagy by modulating the activation of AMPK. In cell models of human breast cancer or in a cyclin D1-deficient model, we observed a cyclin D1-mediated reduction in AMPK activation. Mechanistic investigations showed that cyclin D1 inhibited mitochondrial function, promoted glycolysis, and reduced activation of AMPK (pT172), possibly through a mechanism that involves cyclin D1-Cdk4/Cdk6 phosphorylation of LKB1. Our findings suggest how AMPK activation by cyclin D1 may couple cell proliferation to energy homeostasis