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Understanding the molecular triggers of pancreatic β-cell proliferation may facilitate the development of regenerative therapies for diabetes. Genetic studies have demonstrated an important role for cyclin D2 in β-cell proliferation and mass homeostasis, but its specific function in β-cell division and mechanism of regulation remain unclear. Here, we report that cyclin D2 is present at high levels in the nucleus of quiescent β-cells in vivo. The major regulator of cyclin D2 expression is glucose, acting via glycolysis and calcium channels in the β-cell to control cyclin D2 mRNA levels. Furthermore, cyclin D2 mRNA is down-regulated during S-G(2)-M phases of each β-cell division, via a mechanism that is also affected by glucose metabolism. Thus, glucose metabolism maintains high levels of nuclear cyclin D2 in quiescent β-cells and modulates the down-regulation of cyclin D2 in replicating β-cells. These data challenge the standard model for regulation of cyclin D2 during the cell division cycle and suggest cyclin D2 as a molecular link between glucose levels and β-cell replication.
UbcH5c, a member of the UbcH5 family of protein ubiquitin conjugase E2 enzymes, is a critical component of biological processes in human cells, being the initial ubiquitinating enzyme of substrates like IκB, TP53, and cyclin D1. We report here that the metastasis regulator protein SLUG inhibits the expression of UbcH5c directly through chromatin remodeling and thus, among other downstream effects, elevates the level of cyclin D1, thus enhancing the growth rates of breast cancer cells. Overexpression of SLUG in the SLUG-deficient breast cancer cells significantly decreased the levels of mRNA and protein of UbcH5c but only elevated the protein levels of cyclin D1. On the contrary, knockdown of SLUG in SLUG-high breast cancer cells elevated the levels of UbcH5c while decreasing the level of cyclin D1 protein. SLUG is recruited at the E2-box sequence at the UbcH5c gene promoter along with the corepressor CtBP1 and the effector HDAC1 to silence the expression of this gene. Knockdown of UbcH5c in the SLUG-deficient human breast cells elevated the level of cyclin D1 as well as the rates of proliferation and invasiveness of these cells. Whereas the growth rates of the cells are enhanced due to overexpression of SLUG or knockdown of UbcH5c in the breast cancer cells tested, ER(+) cells also acquire resistance to the anti-estrogen 4-hydroxytamoxifen due to the rise of cyclin D1 levels in these cells. This study thus implicates high levels of SLUG and low levels of UbcH5c as a determinant in the progression of metastatic breast cancer.
The androgen receptor (AR) regulates growth and progression of androgen-dependent as well as androgen-independent prostate cancer cells. Peroxisome proliferator-activated receptor gamma (PPARγ) agonists have been reported to reduce AR activation in androgen-dependent LNCaP prostate cancer cells. To determine whether PPARγ ligands are equally effective at inhibiting AR activity in androgen-independent prostate cancer, we examined the effect of the PPARγ ligands ciglitazone and rosiglitazone on C4-2 cells, an androgen- independent derivative of the LNCaP cell line. Luciferase-based reporter assays and Western blot analysis demonstrated that PPARγ ligand reduced dihydrotestosterone (DHT)-induced increases in AR activity in LNCaP cells. However, in C4-2 cells, these compounds increased DHT-induced AR driven luciferase activity. In addition, ciglitazone did not significantly alter DHT-mediated increases in prostate specific antigen (PSA) protein or mRNA levels within C4-2 cells. siRNA-based experiments demonstrated that the ciglitazone-induced regulation of AR activity observed in C4-2 cells was dependent on the presence of PPARγ. Furthermore, overexpression of the AR corepressor cyclin D1 inhibited the ability of ciglitazone to induce AR luciferase activity in C4-2 cells. Thus, our data suggest that both PPARγ and cyclin D1 levels influence the ability of ciglitazone to differentially regulate AR signaling in androgen-independent C4-2 prostate cancer cells.
Copyright © 2010 Elsevier Inc. All rights reserved.
BACKGROUND - Recent reports have shown that t-DARPP (truncated isoform of DARPP-32) can mediate trastuzumab resistance in breast cancer cell models. In this study, we evaluated expression of t-DARPP in human primary breast tumors, and investigated the role of t-DARPP in regulating growth and proliferation in breast cancer cells.
RESULTS - Quantitative real time RT-PCR analysis using primers specific for t-DARPP demonstrated overexpression of t-DARPP in 36% of breast cancers (13/36) as opposed to absent to very low t-DARPP expression in normal breast tissue (p < 0.05). The mRNA overexpression of t-DARPP was overwhelmingly observed in ductal carcinomas, including invasive ductal carcinomas and intraductal carcinomas, rather than other types of breast cancers. The immunohistochemistry analysis of DARPP-32/t-DARPP protein(s) expression in breast cancer tissue microarray that contained 59 tumors and matched normal tissues when available indicated overexpression in 35.5% of primary breast tumors that were more frequent in invasive ductal carcinomas (43.7%; 21/48). In vitro studies showed that stable overexpression of t-DARPP in MCF-7 cells positively regulated proliferation and anchorage-dependent and -independent growth. Furthermore, this effect was concomitant with induction of phosphorylation of AKT(ser473) and its downstream target phospho(ser9) GSK3β, and increased Cyclin D1 and C-Myc protein levels. The knockdown of endogenous t-DARPP in HCC1569 cells led to a marked decrease in phosphorylation of AKTs(ser473) and GSK3β(ser9). The use of PI3K inhibitor LY294002 or Akt siRNA abrogated the t-DARPP-mediated phosphorylation of AKT(ser473) and led to a significant reduction in cell growth.
CONCLUSIONS - Our findings underscore the potential role of t-DARPP in regulating cell growth and proliferation through PI3 kinase-dependent mechanism.
BACKGROUND/AIM - Cyclin D1 is a mediator of cell-cycle control that is frequently overexpressed in primary ductal breast carcinomas, but its role is controversial. A polymorphism in the CCND1 gene, G870A, results in an aberrantly spliced protein (cyclin D1b) lacking the Thr-286 phosphorylation site necessary for nuclear export. Studies of murine fibroblasts have shown that although overexpression of canonical cyclin D1 (cyclin D1a) alone is not sufficient to drive malignant transformation, expression of nuclear cyclin D1b is oncogenic. Our objectives were to determine whether cyclin D1b is expressed in human breast carcinomas and to characterize the relationship of this protein to both cyclin D1a and clinical outcome in breast cancer patients.
PATIENTS AND METHODS - We performed a prospective cohort study of women with early-stage breast cancer and analyzed cyclin D1a and D1b expression in primary breast tumor sections. Expression was tested for correlation with other breast cancer prognostic factors and clinical outcome, including recurrence or death.
RESULTS - A total of 118 patients were included in this analysis, with a median follow-up of 44 months. Cyclin D1b was expressed in 26% of tumors and cyclin D1a was overexpressed in 27%; co-expression occurred in 4%. Cyclin D1a and/or D1b expression were not significantly associated with estrogen or progesterone receptor negativity, Her2 overexpression, young age, lymph node positivity, high tumor grade, nor large tumor size. The risk of recurrence was higher in those co-expressing D1a and D1b compared to the expression of either alone (relative risk=5.3, 95% confidence interval 1.27 to 22.1, p=0.02). The hazard ratio for those with co-expression compared with those without was 6.05 (p=0.04).
CONCLUSION - Expression of cyclin D1b occurs in primary human breast carcinomas and its coexpression with cyclin D1a may be a marker for increased recurrence risk, independently of other factors.
The Ser/Thr kinase family, RSK, has been implicated in numerous types of hormone-dependent and -independent cancers. However, there has been little consideration of RSKs as downstream mediators of steroid hormone non-genomic effects or of their ability to facilitate steroid receptor-mediated gene expression. Steroid hormone signaling can directly stimulate the MEK/ERK/RSK pathway to regulate cellular proliferation and survival in transformed cells. To date, multiple mechanisms of RSK and steroid hormone receptor-mediated proliferation/survival have been elucidated. For example, RSK enhances proliferation of breast and prostate cancer cells via its ability to control the levels of the estrogen receptor co-activator, cyclin D1. While in lung and other tumors RSK may control apoptosis via estrogen-mediated regulation of mitochondrial integrity. Thus the RSKs could be important anti-cancer therapeutic targets in many different transformed tissues. The recent discovery of RSK-specific inhibitors will advance our current understanding of RSK in transformation and drive these studies into animal and clinical models. In this review we explore the mechanisms associated with RSK in tumorigenesis and their relationship to steroid hormone signaling.
Copyright 2009 Elsevier Inc. All rights reserved.
BACKGROUND - Maintaining the correct balance of proliferation versus differentiation in retinal progenitor cells (RPCs) is essential for proper development of the retina. The cell cycle regulator cyclin D1 is expressed in RPCs, and mice with a targeted null allele at the cyclin D1 locus (Ccnd1-/-) have microphthalmia and hypocellular retinas, the latter phenotype attributed to reduced RPC proliferation and increased photoreceptor cell death during the postnatal period. How cyclin D1 influences RPC behavior, especially during the embryonic period, is unclear.
RESULTS - In this study, we show that embryonic RPCs lacking cyclin D1 progress through the cell cycle at a slower rate and exit the cell cycle at a faster rate. Consistent with enhanced cell cycle exit, the relative proportions of cell types born in the embryonic period, such as retinal ganglion cells and photoreceptor cells, are increased. Unexpectedly, cyclin D1 deficiency decreases the proportions of other early born retinal neurons, namely horizontal cells and specific amacrine cell types. We also found that the laminar positioning of horizontal cells and other cell types is altered in the absence of cyclin D1. Genetically replacing cyclin D1 with cyclin D2 is not efficient at correcting the phenotypes due to the cyclin D1 deficiency, which suggests the D-cyclins are not fully redundant. Replacement with cyclin E or inactivation of cyclin-dependent kinase inhibitor p27Kip1 restores the balance of RPCs and retinal cell types to more normal distributions, which suggests that regulation of the retinoblastoma pathway is an important function for cyclin D1 during embryonic retinal development.
CONCLUSION - Our findings show that cyclin D1 has important roles in RPC cell cycle regulation and retinal histogenesis. The reduction in the RPC population due to a longer cell cycle time and to an enhanced rate of cell cycle exit are likely to be the primary factors driving retinal hypocellularity and altered output of precursor populations in the embryonic Ccnd1-/- retina.
Thiazolidinediones (TZDs) are peroxisome proliferator activated receptor gamma (PPARgamma) ligands that have been reported to reduce proliferation of human prostate cancer cells. However, the mechanisms by which TZDs inhibit prostate cancer cell proliferation are not fully understood. In addition, it is not known if the anti-proliferative effects of TZDs require activation of PPARgamma or are mediated by PPARgamma-independent pathways. The goals of this study were to assess whether TZDs regulate expression of proteins that control the transition from G1 to S phase of the cell cycle and define the role of PPARgamma in these TZD-induced responses in androgen-independent human prostate cancer cell lines. Western blot analysis revealed that growth inhibitory concentrations of the TZDs rosiglitazone and ciglitazone induced expression of the cyclin dependent kinase inhibitor p21 and decreased cyclin D1 levels in the androgen independent PC-3 cell line. Phosphorylation of retinoblastoma protein at Serine 780 was also reduced in PC-3 cells exposed to ciglitazone. Furthermore, growth inhibitory concentrations of ciglitazone increased p21 and lowered cyclin D1 expression within C4-2 cells. PPARgamma-directed siRNAs inhibited the ability of rosiglitazone to regulate expression of cyclin D1 and p21. However, knockdown of PPARgamma did not significantly reduce ciglitazone-induced alterations in cyclin D1 and p21. Furthermore PPARgamma siRNA did not prevent inhibition of PC-3 cell proliferation by either TZD. Thus, activation of PPARgamma is involved in rosiglitazone-induced alterations in cell cycle protein expression. However, the alterations in protein expression and proliferation induced by ciglitazone occur primarily via PPARgamma-independent signaling pathways.
The basal-like subtype of breast cancer is associated with invasiveness, high rates of postsurgical recurrence, and poor prognosis. Aside from inactivation of the BRCA1 tumor-suppressor gene, little is known concerning the mechanisms that cause basal breast cancer or the mechanisms responsible for its invasiveness. Here, we show that the heterogeneous mouse mammary tumor virus-cyclin D1-Cdk2 (MMTV-D1K2) transgenic mouse mammary tumors contain regions of spindle-shaped cells expressing both luminal and myoepithelial markers. Cell lines cultured from these tumors exhibit the same luminal/myoepithelial mixed-lineage phenotype that is associated with human basal-like breast cancer and express a number of myoepithelial markers including cytokeratin 14, P-cadherin, alpha smooth muscle actin, and nestin. The MMTV-D1K2 tumor-derived cell lines form highly invasive tumors when injected into mouse mammary glands. Invasion is associated with E-cadherin localization to the cytoplasm or loss of E-cadherin expression. Cytoplasmic E-cadherin correlates with lack of colony formation in vitro and beta-catenin and p120(ctn) localization to the cytoplasm. The data suggest that the invasiveness of these cell lines results from a combination of factors including mislocalization of E-cadherin, beta-catenin, and p120(ctn) to the cytoplasm. Nestin expression and E-cadherin mislocalization were also observed in human basal-like breast cancer cell lines, suggesting that these results are relevant to human tumors. Together, these results suggest that abnormal Cdk2 activation may contribute to the formation of basal-like breast cancers.
Stress granules aid cell survival in response to environmental stressors by acting as sites of translational repression. We report an unanticipated link between stress granules and the serine/threonine kinase RSK2. In stressed breast cells, endogenous RSK2 colocalizes in granules with TIA-1 and poly(A)-binding protein 1, and the sequestration of RSK2 and TIA-1 exhibits codependency. The RSK2 N-terminal kinase domain controls the direct interaction with the prion-related domain of TIA-1. Silencing RSK2 decreases cell survival in response to stress. Mitogen releases RSK2 from the stress granules and permits its nuclear import via a nucleocytoplasmic shuttling sequence in the C-terminal domain. Nuclear accumulation is dependent on TIA-1. Surprisingly, nuclear localization of RSK2 is sufficient to enhance proliferation through induction of cyclin D1, in the absence of other active signaling pathways. Hence, RSK2 is a pivotal factor linking the stress response to survival and proliferation.