Cyclin D1 Accumulation Research Articles

Thrombin activation of PI3K/PDK1/Akt signaling promotes cyclin D1 upregulation and RPE cell proliferation

The retinal pigment epithelium (RPE) plays an essential role in the survival and function of the neural retina. RPE uncontrolled proliferation leads to the development of proliferative ocular pathologies, among which proliferative vitreoretinopathy (PVR) is the main cause of retinal surgery failure. Upon the breakdown of the BRB due to trauma or metabolic imbalance the contact of RPE with serum-contained thrombin has been shown to stimulate the proliferation of otherwise quiescent RPE cells. Although the molecular mechanisms involved in this effect are still undetermined, thrombin proteolytic activation of protease-activated G protein coupled receptor-1 (PAR-1) activates PI3K and Akt, known to play an essential role in proliferation. The present study demonstrates that: 1) thrombin stimulates Ser 473 Akt phosphorylation without affecting Thr 308 basal phosphorylation in RPE cells; 2) thrombin-induced Akt stimulation promotes cyclin D1 accumulation through the phosphorylation/ inhibition of GSK-3β, thus preventing Thr 286 cyclin D1 phosphorylation, nuclear export and degradation; 3) Akt signaling requires the upstream activation of PI3K and PLC. Since the pharmacological inhibition of these pathways or the silencing of cyclin expression prevent thrombin-induced RPE cell proliferation, these results contribute relevant evidence for establishing the mechanism involved in the development of proliferative eye diseases.

Abstract 2379: Cyclin D3 is important for ErbB2-induced mammary cancer initiation and progression in the absence of Cyclin D1

Abstract The cell cycle regulatory protein Cyclin D1 has been reported to be essential for both normal mammary gland development during pregnancy and the malignant transformation of the mammary epithelium in response to receptor tyrosine kinase (RTK) signaling. We have recently reported that the prolactin receptor-associated kinase Jak2 controls the transcriptional activation and, more importantly, the nuclear accumulation of Cyclin D1 in normal mammary epithelial cells via expression and activation of Akt1. We also demonstrated that the functional ablation of Jak2 and Stat5 in ErbB2-expressing mammary epithelial cells protects against the onset of mammary tumorigenesis. Jak2, however, is not required for the survival and proliferation of neoplastic cells. The constitutive activation of ErbB2 signaling, which is an initial event in the formation of mammary cancer in this model, was able to override the functional role of Jak2 in regulating the expression of Akt1 and Cyclin D1. To further define the role of the downstream effector Cyclin D1 during cancer initiation and progression in response to Jak2 and RTK signaling, we generated an ErbB2-associated mammary cancer model that allows a functional ablation of Cyclin D1 either prior to or following neoplastic transformation. Surprisingly, and in sharp contrast to previous reports, nearly all Cyclin D1-deficient females expressing wildtype ErbB2 developed mammary tumors. While Cyclin D1 deficiency extended the tumor-free survival, this particular cell cycle regulatory protein was clearly not required for tumor maintenance and progression. Growth and proliferation of the vast majority of Cyclin D1-deficient primary mammary cancers was accompanied by an upregulation of Cyclin D3. Since Cyclin D2 expression remained very low, knocking down the last remaining D-type cyclin (i.e. Cyclin D3) in tumor-derived primary cancer cells using three different shRNA constructs was sufficient to attenuate the proliferation rates. Interestingly, the ablation of the pool of all three D-type cyclins did not result in an upregulation of Cyclin E. Collectively, the results of this study show that Cyclin D1 is dispensable for neoplastic transformation of mammary epithelial cells and it might therefore not serve as a sole therapeutic target to treat ErbB2-associated breast cancers as previously suggested. In addition, these observations indicate that Cyclin D1 is most likely not the only downstream effector of Jak2 signaling that mediates the protective effects of a functional ablation of Jak2 in ErbB2-induced mammary carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2379. doi:10.1158/1538-7445.AM2011-2379

Evidence of a Role for Activation of Wnt/β-Catenin Signaling in the Resistance of Plasma Cells to Lenalidomide

Lenalidomide plays an important role in our chemotherapeutic armamentarium against multiple myeloma, in part by exerting direct anti-proliferative and pro-apoptotic effects. Unfortunately, long-term exposure leads to the development of drug resistance through unknown mechanisms, and we therefore sought to identify pathways that could be responsible for this phenotype. Chronic drug exposure produced myeloma cell lines that were tolerant of the direct effects of lenalidomide, with a degree of resistance of up to 2,500-fold. Gene expression profiling and pathway analysis identified dysregulation of the Wnt/β-catenin pathway as a consistent change across four independent cell isolates, and a pair of primary plasma cell samples. Acute drug treatment also increased β-catenin transcription by 3-fold or more, and both acute and chronic exposure resulted in enhanced accumulation of β-catenin protein by up to 20-fold or more. This produced Wnt/β-catenin pathway activation, as judged by increased activity of a lymphoid enhancer factor/T-cell factor promoter reporter, and enhanced accumulation of the downstream targets cyclin D1 and c-Myc. Components of the β-catenin destruction complex were also impacted by lenalidomide, which suppressed casein kinase 1α expression while augmenting glycogen synthase kinase 3α/β phosphorylation. Stimulation of Wnt/β-catenin signaling with recombinant Wnt-3a, or by overexpression of β-catenin, reduced the anti-proliferative activity of lenalidomide. Conversely, suppression of β-catenin with small hairpin RNAs restored plasma cell sensitivity to lenalidomide. Together, these findings support the hypothesis that lenalidomide mediates activation of Wnt/β-catenin signaling in plasma cells as a mechanism of inducible chemoresistance through effects at the transcriptional and post-translational levels.

Translokin (Cep57) Interacts with Cyclin D1 and Prevents Its Nuclear Accumulation in Quiescent Fibroblasts

Nuclear accumulation of cyclin D1 because of altered trafficking or degradation is thought to contribute directly to neoplastic transformation and growth. Mechanisms of cyclin D1 localization in S phase have been studied in detail, but its control during exit from the cell cycle and quiescence is poorly understood. Here we report that translokin (Tlk), a microtubule-associated protein also termed Cep57, interacts with cyclin D1 and controls its nucleocytoplasmic distribution in quiescent cells. Tlk binds to regions of cyclin D1 also involved in binding to cyclin-dependent kinase 4 (Cdk4), and a fraction of cyclin D1 associates to the juxtanuclear Tlk network in the cell. Downregulation of Tlk levels results in undue nuclear accumulation of cyclin D1 and increased Cdk4-dependent phosphorylation of pRB under quiescence conditions. In turn, overexpression of Tlk prevents proper cyclin D1 accumulation in the nucleus of proliferating cells in an interaction-dependent manner, inhibits Cdk4-dependent phosphorylation of pRB and hinders cell cycle progression to S phase. We propose that the Tlk acts as a key negative regulator in the pathway that drives nuclear import of cyclin D1, thus contributing to prevent pRB inactivation and to maintain cellular quiescence.

Ubiquitously Expressed Hematological and Neurological Expressed 1 Downregulates Akt-Mediated GSK3β Signaling, and Its Knockdown Results in Deregulated G2/M Transition in Prostate Cells

As the molecular mechanism of β-catenin deregulation is not well understood, and stabilized β-catenin is known to translocate into the nucleus and activate genes for proliferation, a novel regulatory factor, hematological and neurological expressed 1 (HN1), for Akt-GSK3β-β-catenin axis is reported here. In our studies, HN1 gene structure was characterized. HN1 expression was found to be epidermal growth factor-responsive in PC-3 cells, and protein expression was also upregulated in PC-3 and LNCaP but not in DU145 cells. Additionally, HN1 was found to be downregulated by the specific AKT inhibitor wortmannin but not with PI3K or MAPK inhibitors, LY294002 and PD98059, respectively, in PC-3 and MCF-7 cells. Further, siRNA-mediated knockdown of HN1 resulted in considerable increase in Akt((S473)) and GSK3β((S9),(Y216)) phosphorylations; moreover, subsequent accumulation of β-catenin, increase in c-myc expression, and nuclear accumulation of cyclin D1 were observed in PC-3 cells. Knockdown of HN1 also resulted in prolongation of G(1) phase in cell cycle, increasing tetraploidy, presumably because of cells escaping from abnormal mitosis in PC-3 cells. Consistently, overexpression of HN1 reversed the cell-cycle-specific observations, resulted in accumulation of cells in G(2)/M, and reduced the proliferation rate, which were investigated using flow cytometry and methylthiazol tetrazolium assays. As activating mutations of β-catenin have been demonstrated in late-stage tumors, and β-catenin stabilization was correlated with poor prognosis in previous reports, epidermal growth factor-upregulated HN1 expression might have a role in deregulating the AKT-GSK3β((S9))-mediated signaling as a novel compensating mechanism.

Nuclear Cyclin D1/CDK4 Kinase Regulates CUL4 Expression and Triggers Neoplastic Growth via Activation of the PRMT5 Methyltransferase

Cyclin D1 elicits transcriptional effects through inactivation of the retinoblastoma protein and direct association with transcriptional regulators. The current work reveals a molecular relationship between cyclin D1/CDK4 kinase and protein arginine methyltransferase 5 (PRMT5), an enzyme associated with histone methylation and transcriptional repression. Primary tumors of a mouse lymphoma model exhibit increased PRMT5 methyltransferase activity and histone arginine methylation. Analyses demonstrate that MEP50, a PRMT5 coregulatory factor, is a CDK4 substrate, and phosphorylation increases PRMT5/MEP50 activity. Increased PRMT5 activity mediates key events associated with cyclin D1-dependent neoplastic growth, including CUL4 repression, CDT1 overexpression, and DNA rereplication. Importantly, human cancers harboring mutations in Fbx4, the cyclin D1 E3 ligase, exhibit nuclear cyclin D1 accumulation and increased PRMT5 activity.

Phosphorylation and Ubiquitination of Degron Proximal Residues Are Essential for Class II Transactivator (CIITA) Transactivation and Major Histocompatibility Class II Expression

Major histocompatibility (MHC) class II molecules are cell surface glycoproteins that present extracellular antigens to CD4(+) T cells and are essential for initiation of the adaptive immune response. MHC class II expression requires recruitment of a master regulator, the class II transactivator (CIITA), to the MHC class II promoter. Post-translational modifications to CIITA play important roles in modulating CIITA mediated transcription of various genes in different cell types. We have previously linked regulation of CIITA to the Ubiquitin Proteasome System (UPS), and we and others have demonstrated that mono-ubiquitination of CIITA dramatically increases its transactivity whereas poly-ubiquitination leads to CIITA degradation. Here we identify three degron proximal lysine residues, Lys-315, Lys-330, and Lys-333, and a phosphorylation site, Ser-280, located within the CIITA degron, that regulate CIITA ubiquitination, stability, and MHC class II expression. Together, these findings contribute to the developing post-translational modification code for CIITA.

SENP1 Induces Prostatic Intraepithelial Neoplasia through Multiple Mechanisms

SUMOylation has been shown to modulate DNA replication/repair, cell cycle progression, signal transduction, and the hypoxic response. SUMO (small ubiquitin-like modifier)-specific proteases regulate SUMOylation, but how changes in the expression of these proteases contribute to physiological and/or pathophysiological events remains undefined. Here, we show that SENP1 (sentrin/SUMO-specific protease 1) is highly expressed in human prostate cancer specimens and correlates with hypoxia-inducing factor 1alpha (HIF1alpha) expression. Mechanistic studies in a mouse model indicate that androgen-driven expression of murine SENP1 leads to HIF1alpha stabilization, enhanced vascular endothelial growth factor production, and angiogenesis. Further pathological assessment of the mouse indicates that SENP1 overexpression induces transformation of the normal prostate gland and gradually facilitates the onset of high-grade prostatic intraepithelial neoplasia. Consistent with cell culture studies, SENP1 enhances prostate epithelial cell proliferation via modulating the androgen receptor and cyclin D(1). These results demonstrate that deSUMOylation plays a critical role in prostate pathogenesis through induction of HIF1alpha-dependent angiogenesis and enhanced cell proliferation.

PCB-153 exposure coordinates cell cycle progression and cellular metabolism in human mammary epithelial cells

2,2',4,4',5,5'-Hexachlorobiphenyl (PCB-153) is a non-metabolizable environmental chemical contaminant commonly found in breast milk of PCB exposed individuals, suggesting that chronic exposure to PCB-153 could have adverse health effects. We have shown previously that PCB-153 increased reactive oxygen species levels in non-tumorigenic MCF-10A human mammary epithelial cells, which were associated with DNA damage, growth inhibition, and cytotoxicity. This study investigates the hypothesis that PCB-153 exposure coordinates cell cycle progression and cellular metabolism by inhibiting cyclin D1 accumulation. PCB-153 treated MCF-10A cells exhibited a dose and time dependent decrease in cyclin D1 protein levels. The decrease in cyclin D1 protein levels was associated with an inhibition in AKT and GSK-3beta phosphorylation, which correlated with an increase in cyclin D1-T286 phosphorylation. Fibroblasts carrying a mutant form of cyclin D1 (T286A) were resistant to PCB-153 induced degradation of cyclin D1. Pre-treatment of cells with a proteasome inhibitor (MG132) suppressed PCB-153 induced decrease in cyclin D1 protein levels. Interestingly, suppression in cyclin D1 accumulation was associated with an increase in cellular glucose consumption, and hexokinase II and pyruvate kinase protein levels. These results suggest that cyclin D1 coordinates cell cycle progression and cellular metabolism in PCB-153 treated non-tumorigenic human mammary epithelial cells.

Polychlorinated biphenyl induced ROS signaling delays the entry of quiescent human breast epithelial cells into the proliferative cycle

Polychlorinated biphenyls (PCBs) are environmental chemical contaminants that can produce reactive oxygen species (ROS) by autoxidation of dihydroxy-PCBs and redox-cycling. We investigate the hypothesis that PCB induced perturbations in ROS signaling regulate the entry of quiescent cells into the proliferative cycle. Quiescent MCF-10A human breast epithelial cells were incubated with 0-3 micromolar of 2-(4-chlorophenyl)benzo-1,4-quinone (4-Cl-BQ), 2, 2′, 4, 4′, 5, 5′-hexachlorobiphenyl (PCB 153), and Aroclor 1254 for 4 days. Cells were replated at a lower density and analyzed for cell cycle phase distributions, ROS levels, MnSOD expression, and cyclin D1 protein levels. Quiescent cells incubated with 4-Cl-BQ showed the maximal delay in entering S phase. This delay was associated with a decrease in MnSOD activity, protein and mRNA levels, and an increase in cellular ROS levels. Results from the mRNA turnover assay showed that the 4-Cl-BQ treatment selectively enhanced the degradation of the 4.2 kb MnSOD transcript, while the half-life of the 1.5 kb transcript did not change. Accumulation of cyclin D1 protein levels in replated cells was suppressed in cells treated with 4-Cl-BQ. Pretreatment of quiescent cells with polyethylene glycol-conjugated superoxide dismutase and catalase suppressed 4-Cl-BQ induced increase in ROS levels, which was consistent with an increase in cyclin D1 accumulation, and entry into S phase. These results showed 4-Cl-BQ induced perturbations in ROS signaling inhibit the entry of quiescent cells into S phase.

The HTLV-1 tax protein cooperates with phosphorylated CREB, TORC2 and p300 to activate CRE-dependent cyclin D1 transcription

Adult T-cell leukemia/lymphoma is a fatal malignancy etiologically linked to infection with the human T-cell leukemia virus (HTLV-1). The virally-encoded oncoprotein Tax activates transcription of HTLV-1 and cellular genes by cooperating with cellular transcription factors. Cyclin D1 is a pivotal regulator of cell cycle progression, and increased expression strongly correlates with malignant transformation. Here, we characterize the mechanism of Tax transactivation of cyclin D1. We find that cyclin D1 transcript levels are elevated in HTLV-1 infected cells and that Tax physically associates with the cyclin D1 gene in vivo. Tax binds the cyclin D1 promoter-proximal cyclic AMP response element (CRE) in the presence of phosphorylated CREB (pCREB) in vitro, and together the Tax/pCREB complex recruits the cellular coactivator p300 to the promoter via this unconventional Tax-responsive element. We further show that Transducer of Regulated CREB 2 (TORC2) cooperates with Tax to further enhance p300 recruitment to the cyclin D1 promoter in vitro, consistent with enhanced cyclin D1 expression in the presence of Tax and TORC2. Together, our findings support a model in which Tax-induced accumulation of cyclin D1 shortens the G1 phase of the cell cycle, promotes mitotic replication of the virus, and drives selection and expansion of malignant T-cells.

ON 01910.Na Suppresses Cyclin D1 Accumulation in trisomy 8 Myelodysplastic Syndromes Patients While Decreasing Bone Marrow CD34+ Blast Counts and Aneuploid Clone Size.

Abstract 120Patients with high risk MDS can be successfully treated with 5-azacytidine, or with lenalidomide but non-responding patients have few treatment options. Chemotherapy produces significant morbidity and very short remissions and most patients are too old for bone marrow transplantation. We previously demonstrated up-regulation of c-myc, survivin, and cyclin D1 in CD34+ cells in patients with trisomy 8 (and selected patients with monosomy 7). siRNA-mediated knockdown of survivin or c-myc decreased trisomy 8 cell growth in vitro (Sloand et al, Blood 2007, 110: 822). We postulated that increased cyclin D1 causes upregulation of survivin, resulting in resistance of these cells to apoptosis. The styryl sulfone, ON 01910.Na, decreases cyclin D1 accumulation in cultured bone marrow from patients with high risk trisomy 8 MDS and in some monosomy 7 patients (who also show upregulation of cyclin D1), while selectively decreasing blasts and aneuploidy with this cytogenetic abnormality (ASH Abstracts Nov 2008; 112: 1651). Here we examine the clinical response to ON1910 in an ongoing phase I/II clinical trial in which 13 evaluable patients with intermediate-1(int-1) to high risk MDS and treatment-refractory trisomy 8 AML were enrolled. Patients were treated with escalating doses of ON 01910.Na at 800 mg/m2 × 2 days every 3/4 weeks, 800 mg/m2 × 3 days every 2 weeks, 800 mg/m2 × 5 days every 2 weeks, and 1500 mg/m2 × 2 days every 3/4 weeks at two institutions. No significant toxicity could be ascribed to the drug. Patients with trisomy 8 and monosomy 7 demonstrated significant declines in aneuploidy measured by florescence in situ hybridization (FISH) (mean aneuploidy; 50% before and 24% after 1 cycle of treatment; p=0.02 :Fig below). Rather than becoming cytopenic, many patients showed substantial improvements of blood counts and one patient (01-02; graphic shown below) became red cell transfusion-independent and maintains his remission 14 months after stopping therapy. Cyclin D1 measurement by flow cytometry showed decreases of this protein in both CD34 and CD33 cells during infusion of ON 1910 infusion (example shown in Fig below). Results from individual evaluable patients are shown in table 1. These results indicate that modulation of cell cycle control by cyclin D1 may represent a novel targeted approach for trisomy 8 and monosomy 7 MDS. [Display omitted] Table 1ProtocolPIDON 01910.NaIPSS Pre-TrtWeeks on studyCytogeneticsPrior 5-Aza% Blasts BMHISurvival weeksDosage mg/m2/24hPre-RxWeek 4-8 F-Up07-H-022501-01800/72h Q2WHigh10+8, -7, 5q-Yes174N,P1901-02800/72h Q2WHigh16−7No52E56+01-03800/72h Q2WHigh8−7, +8No105N52+02-01800/120h Q2WHigh6+8No5019P,E2602-02*800/120h Q2WHigh5+8/ComplexYes15133302-03800/120h Q1WAML2+8No80103402-04800/120h Q2WHigh14t (3,21)No51N21+02-05800/120h Q2WAML2+8 complexNo30ND202-06800/120h Q2WHigh4++11No6624+04-1501-04800/48h 3/4 wksHigh19++14Yes2530N19+01-08800/48h 3/4 wksHigh17+ComplexNo251117+01-09800/48h 3/4 wksInt-117+NormalNo1215E17+01-111500/48h 3/4 wksInt-215+5q-/7 abnNo9515+01-121500/48h 3/4 wksInt-115+NDNo10215+01-131500/48h 3/4 wksInt-210+8/+13Yes152013BM=Bone Marrow; h=hours; wks=weeks; Q2W=every other week; SD=Stable disease; PD=Progressive disease; HI=Hematological Improvement (P=Platelet; E=Erythroid; N=Neutrophil): ND not done*Patient did not complete two infusions because of access problems Disclosures:Sloand:Onconova: Research Funding. Olnes:Onconova: Research Funding. Galili:Onconova: Research Funding. Wilhelm:Onconova: Employment. Groopman:Onconova: Membership on an entity's Board of Directors or advisory committees. Raza:Onconova: Research Funding.

Cyclin D1 Induction by Benzo[a]pyrene-7,8-diol-9,10-epoxide via the Phosphatidylinositol 3-Kinase/Akt/MAPK- and p70s6k-dependent Pathway Promotes Cell Transformation and Tumorigenesis

Benzo[a]pyrene-7,8-diol-9,10-epoxide (B[a]PDE), the major metabolite of B[a]P, has been well recognized as one ubiquitous carcinogen, but the molecular mechanism involved in its carcinogenic effect remains obscure. In the present study, we found that bronchial epithelial cells (Beas-2B) and hepatocytes treated with B[a]PDE presented a significant increase of cyclin D1 expression. Moreover, Akt, p70(s6k), and MAPKs including JNK, Erks, and p38 were notably activated in B[a]PDE-treated Beas-2B cells, whereas NF-kappaB, NFAT, and Egr-1 were not. Our results demonstrated that JNK and Erks were required in B[a]PDE-induced cyclin D1 expression because the inhibition of JNK or Erks by a selective chemical inhibitor or dominant negative mutant robustly impaired the cyclin D1 induction by B[a]PDE. Furthermore, we found that overexpression of the dominant negative mutant of p85 (regulatory subunit of phosphatidylinositol 3-kinase) or Akt dramatically suppressed B[a]PDE-induced JNK and Erk activation as well as cyclin D1 expression, suggesting that cyclin D1 induction by B[a]PDE is via the phosphatidylinositol 3-kinase/Akt/MAPK-dependent pathway. In addition, we clarified that p70(s6k) is also involved in B[a]PDE-induced cyclin D1 expression because rampamycin pretreatment dramatically reduced cyclin D1 induction by B[a]PDE. More importantly, we demonstrated that up-regulated cyclin D1 by B[a]PDE plays a critical role in oncogenic transformation and tumorigenesis of Beas-2B cells. These results not only broaden our knowledge of the molecular mechanism of B[a]PDE carcinogenicity but also lead to the further study of chemoprevention of B[a]PDE-associated human cancers.

Lysine 269 is essential for cyclin D1 ubiquitylation by the SCFFbx4/αB-crystallin ligase and subsequent proteasome-dependent degradation

Protein ubiquitylation is a complex enzymatic process that results in the covalent attachment of ubiquitin, via Gly-76 of ubiquitin, to an ε-NH2-group of an internal lysine residue in a given substrate. While E3 ligases frequently utilize lysines adjacent to the degron within the substrate, many substrates can be targeted to the proteasome via polyubiquitylation of any lysine. We have assessed the role of lysine residues proximal to the cyclin D1 phosphodegron for ubiquitylation by the SCFFbx4/αB-crystallin ubiquitin ligase and subsequent proteasome-dependent degradation of cyclin D1. The work described herein reveals a requisite role for Lys-269 (K269) for the rapid, poly-ubiquitin mediated degradation of cyclin D1. Mutation of lysine 269, which is proximal to the phosphodegron sequence surrounding Thr-286 in cyclin D1, not only stabilizes cyclin D1, but also triggers cyclin D1 accumulation within the nucleus thereby promoting cell transformation. In addition, D1-K269R is resistant to genotoxic stress induced degradation, similar to non-phosphorylatable D1-T286A, supporting the critical role for the post-translational regulation of cyclin D1 in the response to DNA damaging agents. Strikingly, while mutation of lysine 269 to arginine inhibits cyclin D1 degradation, it does not inhibit cyclin D1 ubiquitylation in vivo demonstrating that ubiquitylation of a specific lysine can influence substrate targeting to the 26S proteasome.

The Absence of Caveolin-1 Increases Proliferation and Anchorage- Independent Growth by a Rac-Dependent, Erk-Independent Mechanism

Anchorage-independent growth (AIG) of cancer cells requires escape from integrin-mediated signals. A protein frequently downregulated in cancer, caveolin-1 (Cav1), mediates integrin control of several growth-regulatory pathways. We report that loss of Cav1 results in faster exit from quiescence and progress through the cell cycle, proliferation without anchorage to substrate, and absence of cyclin D1 downregulation upon serum deprivation or detachment. Surprisingly, this proliferative advantage is independent of Erk-mitogen-activated protein kinase signaling; instead, cyclin expression and cell cycle progression in the absence of Cav1 are driven by increased membrane order and Rac targeting. AIG was induced in Cav1-expressing cells by forced membrane targeting of Rac1 or by inhibiting Cav1-mediated internalization of plasma membrane ordered domains at which Rac1 accumulates. Restoring Rho activity, which is downregulated after loss of Cav1, antagonizes Rac1 and prevents cyclin D1 accumulation after serum starvation or loss of adhesion. Anchorage independence and increased proliferation in Cav1-deficient tumoral and null cells are thus due to an increased fraction of active Rac1 at membrane ordered domains. These results provide insight into the mechanisms regulating growth of cancer cells, which frequently lose Cav1 function.

Targeting Janus Kinase 2 in Her2/neu-Expressing Mammary Cancer: Implications for Cancer Prevention and Therapy

The Janus kinase 2 (Jak2) is essential for normal mammary gland development, but this tyrosine kinase and its main effector, signal transducer and activator of transcription 5, are also active in a significant subset of human breast cancers. We have recently reported that Jak2 controls the expression and nuclear accumulation of cyclin D1. Because this particular D-type cyclin has been suggested to be a key mediator for ErbB2-associated mammary tumorigenesis, we deleted Jak2 from ErbB2-expressing mammary epithelial cells prior to tumor onset and in neoplastic cells to address whether this tyrosine kinase plays a role in the initiation as well as progression of mammary cancer. Similar to cyclin D1-deficient mice, the functional ablation of Jak2 protects against the onset of mammary tumorigenesis. In contrast, the deletion of Jak2 from neoplastic cells or the acute, ligand-inducible down-regulation of this tyrosine kinase in an orthotopic transplant model did not affect the growth and survival of cancer cells. The constitutive activation of ErbB2 signaling, which is an initial event in the formation of mammary cancer, was able to override the functional role of Jak2 in regulating the expression of Akt1 and cyclin D1. This might be a compensatory mechanism that explains why Jak2 is a relevant target for preventing the initiation but not the progression of ErbB2-associated mammary cancer.

Znaczenie prognostyczne ekspresji cykliny D1 i cykliny B u pacjentów z rakiem krtani leczonych radioterapią

Cyclin B1 and cyclin D1 playing the role of regulatory subunits of cyclin-dependent kinases responsible for progression of cell cycle are involved in carcinogenesis of head and neck tumors. The aim of this study is to investigate their value for predicting clinical outcome after radiotherapy of laryngeal squamous cell cancer (LSCC). The study included 50 patients with LSCC treated between the years 1998 and 2003 with radiotherapy. Tissue samples were studied immunohistochemically for detection of cyclin B1 and cyclin D1 and their expression were correlated with clinicopathological factors, local tumor control and patient survival. Accumulation of cyclin D1 and cyclin B1 were detected in 36% and 48% of tumors respectively. No relationship was observed between immunostaining for analyzed proteins and clinicopathologic factors. Cyclin B1 overexpression was associated with higher rate of locoregional recurrence. Five-year disease free survival rate for patients with cyclin B1 positive tumors was 73% v. 84% for cyclin B1 negative patients (p=0.005). The expression of cyclin D1 in LSCC does not seem to have a prognostic significance. Overexpression of cyclin B1 may be an indicator of the risk of locoregional recurrence in patient receiving radiotherapy. Thus cyclin B1 detected by immunohistochemistry can be useful for predicting outcome of radiotherapy in patients with LSCC.

Altered expression of key cell cycle regulators in renal cell carcinoma associated with Xp11.2 translocation

Renal cell carcinoma (RCC) is a rare tumor in the pediatric population. Recently, a phenotypically and genetically distinct kidney carcinoma, mainly prevalent in children and associated with an Xp11.2 translocation or TFE3 gene fusion, has been described. It has been advanced that in this subtype of RCC, there is an accumulation of cyclin D1, cyclin D3, and p21 (wafl/cip1). The aim of the present study was to figure out in two pediatric RCC recently diagnosed in our department (one clear cell-type RCC and one TFE3-positive RCC) whether those features are indeed specific of the latter tumor or occur in pediatric RCC irrespective of the tumor type. The following immunostains were performed in both cases: Ki67, p16 ink4a, p21 (wafl/cip1), p27 kip1, p53, p63, mdm2, cyclin D1, cyclin D3, TFE3, CD10, vimentin, E-cadherin, and RCC-antigen. We observed in the TFE3-positive carcinoma an intense immunoreaction for p21 (wafl/cip1), cyclin D1, and cyclin D3, without expression for p53, p16, p27 kip1, and mdm2, whereas the immunoexpression profile observed in the classic RCC was similar to that of clear cell, adult-type RCC. Our study confirms that TFE3-positive RCC exhibits a deregulation of the cell cycle apparently unrelated to the young age of the patients.

Aberrant CpG Island Hypermethylation of RASSF1A in Gastric Cardia Adenocarcinoma

Ras-association domain family 1A (RASSF1A) gene, a candidate tumor suppressor gene, is inactivated in several human tumors, usually by hypermethylation of its promoter region. RASSF1A induces cell cycle arrest through inhibition of cyclin D1 accumulation. In this work, the promoter methylation status of the RASSF1A in 92 gastric cardia adenocarcinoma (GCA) and corresponding normal tissues were investigated using Methylation-specific PCR (MSP) approach, immunohistochemistry method and RT-PCR were used respectively to examine the protein expression and mRNA expression of RASSF1A in tumors and corresponding normal tissues. Cyclin D1 expression was examined by immunohistochemistry. RASSF1A was methylated in 54/92 (58.7%) tumor specimens, which was significantly higher than that in corresponding normal tissues (p <.001). Methylation frequencies of stage III and IV tumor tissues were significantly higher than that in stage I and II tumor tissues (p <.05). By immunostaining, 43/92 (46.7%) tumor tissues demonstrated heterogeneous, positive immunostaining of tumor tissues was significantly reduced with comparison to matched normal tissues (p <.001). mRNA expressions of RASSF1A in GCA tumor tissues were reduced significantly with comparison to the corresponding normal tissues (OD value: 0.2376 ± 0.2315 vs 0.6874 ± 0.2668, p <.001). RASSF1A mRNA expression in methylation group of GCA was significantly different from that in unmethylation group (p <.001). Cyclin D1 hyper-expression was found in 72/92 (78.3%) cases and correlated with RASSF1A methylation (p <.05). Our data suggested that epigenetic silencing of RASSF1A gene expression by promoter hypermethylation may play an important role in GCA.

Cyclin D1 repressor domain mediates proliferation and survival in prostate cancer

Regulation of the androgen receptor (AR) is critical to prostate cancer (PCa) development; therefore, AR is the first line therapeutic target for disseminated tumors. Cell cycle dependent accumulation of cyclin D1 negatively modulates the transcriptional regulation of the AR through discrete, CDK4-independent mechanisms. The transcriptional co-repressor function of cyclin D1 resides within a defined motif termed ther repressor domain (RD), and it was hypothesized that this motif could be utilized as a platform to develop new strategies for blocking AR function. Here, we demonstrate that expression of the RD peptide is sufficient to disrupt AR transcriptional activation of multiple, prostate-specific AR target genes. Importantly, these actions are sufficient to specifically inhibit S-phase progression in AR-positive PCa cells, but not in AR-negative cells or tested AR-positive cells of other lineages. As expected, impaired cell cycle progression resulted in a suppression of cell doubling. Additionally, cell death was observed in AR-positive cells that maintain androgen dependence and in a subset of castrate-resistant PCa cells, dependent on Akt activation status. Lastly, the ability of RD to cooperate with existing hormone therapies was examined, which revealed that RD enhanced the cellular response to an AR antagonist. Together, these data demonstrate that RD is sufficient to disrupt AR-dependent transcriptional and proliferative responses in PCa, and can enhance efficacy of AR antagonists, thus establishing the impetus for development of RD-based mimetics.