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1.  siRNA-Based Targeting of Cyclin E Overexpression Inhibits Breast Cancer Cell Growth and Suppresses Tumor Development in Breast Cancer Mouse Model 
PLoS ONE  2010;5(9):e12860.
Cyclin E is aberrantly expressed in many types of cancer including breast cancer. High levels of the full length as well as the low molecular weight isoforms of cyclin E are associated with poor prognosis of breast cancer patients. Notably, cyclin E overexpression is also correlated with triple-negative basal-like breast cancers, which lack specific therapeutic targets. In this study, we used siRNA to target cyclin E overexpression and assessed its ability to suppress breast cancer growth in nude mice. Our results revealed that cyclin E siRNA could effectively inhibit overexpression of both full length and low molecular weight isoforms of cyclin E. We found that depletion of cyclin E promoted apoptosis of cyclin E-overexpressing cells and blocked their proliferation and transformation phenotypes. Significantly, we further demonstrated that administration of cyclin E siRNA could inhibit breast tumor growth in nude mice. In addition, we found that cyclin E siRNA synergistically enhanced the cell killing effects of doxorubicin in cell culture and this combination greatly suppressed the tumor growth in mice. In conclusion, our results indicate that cyclin E, which is overexpressed in 30% of breast cancer, may serve as a novel and effective therapeutic target. More importantly, our study clearly demonstrates a very promising therapeutic potential of cyclin E siRNA for treating the cyclin E-overexpressing breast cancers, including the very malignant triple-negative breast cancers.
doi:10.1371/journal.pone.0012860
PMCID: PMC2942895  PMID: 20877462
2.  Cdk2 is required for breast cancer mediated by the low molecular weight isoform of cyclin E 
Cancer research  2011;71(9):3377-3386.
Cyclin E activates Cdk2, controls centrosome duplication and regulates histone gene transcription. Cyclin E is deregulated in cancer and appears as low molecular weight (LMW) isoforms that correlate strongly with decreased survival in breast cancer patients. Transgenic mice overexpressing LMW cyclin E have increased incidence of mammary tumors and distant metastasis when compared to full length cyclin E. To specifically test the requirement for Cdk2 in LMW-cyclin E mediated mammary tumorigenesis, we generated transgenic mice, which expressed LMW-cyclin E in a Cdk2 deficient background. We found that mammary gland development proceeds relatively normally in these animals, indicating that Cdk2 kinase activity is largely dispensable for this process. However, Cdk2 deficient mice were completely resistant to LMW-cyclin E mediated mammary tumors. Cdk2 wild-type or heterozygous mice succumbed to mammary tumors with mean latencies of 16 and 19.5 months, respectively, but Cdk2 nullizygous littermates did not display tumors through 24 months. Similarly, continuous administration of two different Cdk inhibitors significantly delayed LMW-cyclin E induced mammary tumor progression. Triple transgenic mice generated in a p53 heterozygous background also displayed no tumors. We also found that Cdk2 silencing induced cell death in LMW-overexpressing breast cancer cell lines, but not in cell lines lacking LMW expression. Our findings establish a requirement for Cdk2 in LMW-cyclin E mediated mammary tumorigenesis, arguing that human breast tumors overexpressing LMW-cyclin E are prime candidates for anti-Cdk2 therapy.
doi:10.1158/0008-5472.CAN-10-4086
PMCID: PMC3085722  PMID: 21385896
Low molecular weight cyclin E; Cdk2; transgenic mice; breast cancer; roscovitine; meriolin
3.  Chromosomal instability and lack of cyclin E regulation in hCdc4 mutant human breast cancer cells 
Breast Cancer Research  2004;6(5):R531-R539.
Introduction
Cyclin E, a G1 cyclin essential for G1–S phase transition, is known to have a profound effect on tumorigenesis. Elevated levels of cyclin E have been associated with breast cancer, and chromosomal instability observed in breast cancer is suggested to be associated with constitutive expression of cyclin E. It was previously demonstrated that SUM149PT human breast cancer cells show very high levels of cyclin E expression by western analysis and that they express a nonfunctional cyclin E ubiquitin ligase due to a mutation in the F-box protein hCdc4.
Methods
We examined cyclin E expression in both MCF10A and SUM149PT cells using western blot analysis and flow cytometry. Immunofluorescence was utilized for the localization of cyclin E in both normal and breast cancer cells. In addition, array comparative genomic hybridization analysis was performed to compare chromosome copy number alterations with levels of cyclin E expression among a panel of breast cancer cell lines.
Results
SUM149PT cells overexpress cyclin E on a cell per cell basis for the duration of the cell cycle. High cyclin E levels are maintained throughout the S phase, and SUM149PT cells exhibit an S phase delay or arrest probably due to cyclin E overexpression. In addition, comparative genomic hybridization indicated that SUM149PT cells exhibit many chromosome copy number alterations, which may reflect prior or ongoing genomic instability. However, no direct correlation was observed between cyclin E levels and genomic copy number alteration in a panel of human breast cancer cell lines.
Conclusions
Cyclin E is overexpressed at high levels throughout the cell cycle in SUM149PT cells, which is in stark contrast to cyclin E degradation observed in the mid to late S phase of normal cells. SUM149PT cells are unable to regulate cyclin E and also exhibit many copy number alterations. However, there was a lack of direct correlation between cyclin E overexpression and chromosomal instability across a panel of other breast cancer cell lines examined.
doi:10.1186/bcr900
PMCID: PMC549168  PMID: 15318934
breast cancer; cyclin E; genomic instability
4.  Galectin-3 and cyclin D1 expression in non-small cell lung cancer 
Introduction
Lung cancer is a major cause of mortality and morbidity worldwide. Galectin-3 is multifunctional protein, which is involved in regulation of cell growth, cell adhesion, cell proliferation, angiogenesis and apoptosis. Cyclin D1 together with other cyclin plays an important role in cell cycle control. Cyclin D1 regulates the G1-to-S phase transition. The aim of this study was the evaluation of correlations between clinicopathological findings and cyclin D1 and galectin-3 expression in non-small cell lung cancer (NSCLC). We wanted also to analyze the prognostic value of cyclin D1 and galectin-3 expression. Moreover we tried to evaluate the correlations between galectin-3 and cyclin D1 expression in tumor tissue.
Materials and methods
We used the immunochemistry method to investigate the expression of galectin-3 and cyclin D1 in the paraffin-embedded tumor tissue of 47 patients (32 men and 15 women; mean age 59.34 ± 8.90). years. We used monoclonal antibodies to cyclin D1 (NCL-L-cyclin D1-GM clone P2D11F11 NOVO CASTRA) and to galectin-3 (mouse monoclonal antibody NCL-GAL3 NOVO CASTRA).
Results
Galectin-3 expression was positive in 18 cases (38.29%) and cyclin D1 in 39 (82.97%). We showed only weak trend, that galectin-3 expression was lower in patients without lymph node involvement (p = 0.07) and cyclin D1 expression was higher in this group (p = 0.080). We didn't reveal differences in cyclin D1 and galectin-3 expression in SCC and adenocarcinoma patients. We didn't demonstrated also differences in galectin-3 and cyclin D1 expression depending on disease stage. Moreover we analyzed the prognostic value of cyclin D1 expression and galectin-3 in all examinated patients and separately in SCC and in adenocarcinoma and in all stages, but we didn't find any statistical differences. We demonstrated that in galectin-3 positive tumors cyclin D1 expression was higher (96.55% vs 61.11%, Chi2 Yatesa 7.53, p = 0.0061) and we revealed negative correlation between cyclin D1 and galectin-3 expression (R Spearman -0.458, p = 0.0011). In squamous cell lung cancer we didn't observed correlations between these both examinated markers (R = -0.158, p = 0.460), and in adenocarcinoma the negative correlation was very strong (R = -0.829 p = 0.000132).
Conclusions
We didn't reveal any important correlations between clinicopathological findings and galectin-3 and cyclin D1 expression and in non small cell lung cancer. We didn't observed also prognostic value of cyclin D1 or galectin-3 expression. But we showed higher cyclin D1 expression in galectin-3 negative tumor tissues. We revealed also differences in correlations between galectin-3 and cyclin D1 expression in two main histopathological types of NSCLC.
doi:10.1186/1756-9966-30-101
PMCID: PMC3214148  PMID: 22024187
galectin-3; cyclin D1; non-small cell lung cancer; prognostic factor
5.  Leptin regulates cyclin D1 in luminal epithelial cells of mouse MMTV-Wnt-1 mammary tumors 
Purpose
Leptin, an adipose secreted cytokine, is implicated in mammary cancer stem cell self-renewal and tumor growth in Murine Mammary Tumor Virus (MMTV)-Wnt-1 transgenic mice. In vitro studies indicate that leptin induces expression of Cyclin D1, a cell-cycle control protein necessary for mammary tumor development. The aim of the present study was to assess Cyclin D1 expression in spontaneous tumors that develop in the MMTV-Wnt-1 transgenic mice and interrogate the in vivo effect of leptin.
Materials and methods
Cells derived from spontaneous MMTV-Wnt-1 tumors were orthotopically transplanted into wild type, leptin-deficient, and hyperleptinemic mice. After 6 weeks, the tumors were collected and formalin fixed. Immunoflurescence staining was used to assess Cyclin D1, keratin 8, α-SMA, phospho-AKT expression.
Results
Cyclin D1 is expressed exclusively in luminal keratin 8 immunoreactive tumor cells and is dependent on the adipose secreted hormone leptin. Tumor cell transplant into leptin-deficient mice resulted in approximately an 80% reduction of Cyclin D1 immunoreactivity in keratin 8 cells and this was independent of Akt activation.
Conclusions
Collectively, these data and our previous findings indicate that inhibition of leptin signaling provides an excellent therapeutic target for breast cancer. The current data indicate that in luminal mammary tumors, leptin antagonists would potentially inhibit growth in a Cyclin D1-dependent mechanism. In contrast, in basal mammary tumors, leptin antagonists would inhibit growth in an Akt-dependent manner leading to reduction in CSC self-renewal. Thus, leptin therapeutics may inhibit breast cancer via distinct mechanisms dependent on the tumor type.
doi:10.1007/s00432-012-1252-9
PMCID: PMC3520612  PMID: 22692856
6.  Multiple Cellular Mechanisms Related to Cyclin A1 in Prostate Cancer Invasion and Metastasis 
Background
Cyclin A1 is a cell cycle regulator that has been implicated in the progression of prostate cancer. Its role in invasion and metastasis of this disease has not been characterized.
Methods
Immunohistochemistry and cDNA microarray analyses were used to assess protein and mRNA expression of cyclin A1 and proteins with roles in metastasis, including vascular endothelial growth factor (VEGF), metalloproteinase 2 (MMP2), and MMP9, in human prostate cancer. Transient transfection and infection with viral vectors expressing cyclin A1 and short hairpin RNA (shRNA) targeting cyclin A1 were used to study the effects of altered cyclin A1 expression in PC3 prostate cancer cells. The BrdU assay, annexin V staining, and invasion chambers were used to examine cyclin A1 effects on proliferation, apoptosis, and invasion, respectively. The role of cyclin A1 and androgen receptor (AR) in transcription of VEGF and MMP2 was assessed by promoter mutation and chromatin immunoprecipitation. The effect of cyclin A1 expression on tumor growth and metastasis was analyzed in a mouse model of metastasis. All statistical tests were two-sided.
Results
Cyclin A1 protein and mRNA expression were statistically significantly higher in prostate cancers than in adjacent benign tissues. A statistically significant correlation between expression of cyclin A1 and of MMP2, MMP9, and VEGF was observed in prostate tumors from 482 patients (P values from Spearman rank correlation tests < .001). PC3 cells that overexpressed cyclin A1 showed increased invasiveness, and inhibition of cyclin A1 expression via shRNA expression reduced invasiveness of these cells. Eight of 10 mice (80%) bearing PC3 cells overexpressing cyclin A1 had infiltration of tumor cells in lymph node, liver, and lung, but all 10 mice bearing tumors expressing control vector were free of liver and lung metastases and only one mouse from this group had lymph node metastasis (P values from Fisher exact tests < .001). Cyclin A1, in concert with AR, bound to and increased expression from the VEGF and MMP2 promoters.
Conclusions
Cyclin A1 contributes to prostate cancer invasion by modulating the expression of MMPs and VEGF and by interacting with AR.
doi:10.1093/jnci/djn214
PMCID: PMC2467435  PMID: 18612129
7.  Combined effect of cyclin D3 expression and abrogation of cyclin D1 prevent mouse skin tumor development 
Cell Cycle  2012;11(2):335-342.
We have previously demonstrated that ras-mediated skin tumorigenesis depends on signaling pathways that act preferentially through cyclin D1 and D2. Interestingly, the expression of cyclin D3 inhibits skin tumor development, an observation that conflicts with the oncogenic role of D-type cyclins in the mouse epidermis. Here, we show that simultaneous up and downregulation of particular members of the D-type cyclin family is a valuable approach to reduce skin tumorigenesis. We developed the K5D3/cyclin D1−/− compound mouse, which overexpresses cyclin D3 but lacks expression of cyclin D1 in the skin. Similar to K5D3 transgenic mice, keratinocytes from K5D3/cyclin D1−/− compound mice show a significant reduction of cyclin D2 levels. Therefore, this model allows us to determine the effect of cyclin D3 expression when combined with reduced or absent expression of the remaining two members of the D-type cyclin family in mouse epidermis. Our data show that induced expression of cyclin D3 compensates for the reduced level of cyclin D1 and D2, resulting in normal keratinocyte proliferation. However, simultaneous ablation of cyclin D1 and downregulation of cyclin D2 via cyclin D3 expression resulted in a robust reduction in ras-mediated skin tumorigenesis. We conclude that modulation of the levels of particular members of the D-type cyclin family could be useful to inhibit tumor development and, in particular, ras-mediated tumorigenesis.
doi:10.4161/cc.11.2.18774
PMCID: PMC3293382  PMID: 22214766
cell cycle; D-type cyclins; skin; carcinogenesis; epidermis
8.  Cyclin A Degradation by Primate Cytomegalovirus Protein pUL21a Counters Its Innate Restriction of Virus Replication 
PLoS Pathogens  2013;9(12):e1003825.
Cyclin A is critical for cellular DNA synthesis and S phase progression of the cell cycle. Human cytomegalovirus (HCMV) can reduce cyclin A levels and block cellular DNA synthesis, and cyclin A overexpression can repress HCMV replication. This interaction has only been previously observed in HCMV as murine CMV does not downregulate cyclin A, and the responsible viral factor has not been identified. We previously reported that the HCMV protein pUL21a disrupted the anaphase-promoting complex (APC), but a point mutant abrogating this activity did not phenocopy a UL21a-deficient virus, suggesting that pUL21a has an additional function. Here we identified a conserved arginine-x-leucine (RxL) cyclin-binding domain within pUL21a, which allowed pUL21a to interact with cyclin A and target it for proteasome degradation. Homologous pUL21a proteins from both chimpanzee and rhesus CMVs also contained the RxL domain and similarly degraded cyclin A, indicating that this function is conserved in primate CMVs. The RxL point mutation disabled the virus' ability to block cellular DNA synthesis and resulted in a growth defect similar to pUL21a-deficient virus. Importantly, knockdown of cyclin A rescued growth of UL21a-deficient virus. Together, these data show that during evolution, the pUL21a family proteins of primate CMVs have acquired a cyclin-binding domain that targets cyclin A for degradation, thus neutralizing its restriction on virus replication. Finally, the combined proteasome-dependent degradation of pUL21a and its cellular targets suggests that pUL21a may act as a novel suicide protein, targeting its protein cargos for destruction.
Author Summary
Cyclins are evolutionarily conserved proteins that associate with cyclin-dependent kinases (CDKs) to regulate phosphorylation of multiple substrates to promote cell-cycle progression. Many viruses manipulate the cell cycle in order to create an environment suitable for replication; however, only few examples exist where viruses modulate cyclin activity. Here, we identified a cyclin-binding domain within the human cytomegalovirus (HCMV) protein pUL21a that confers its ability to interact with cyclin A and target it for proteasome degradation. Cyclin A promotes cellular DNA replication, which consumes important enzymes and metabolites needed for viral replication, making it important for large viruses like HCMV to block this protein's activity. In accord, the ability of pUL21a to degrade cyclin A was necessary for the virus to block cellular DNA replication and promote viral replication. Importantly, ablating cyclin A expression restored replication to a virus lacking pUL21a, demonstrating that cyclin A has the intrinsic ability to restrict viral replication, but is specifically countered by pUL21a. Together with our previous work showing that pUL21a also regulates the anaphase-promoting complex, another master cell cycle regulator, our studies have now revealed that HCMV has elegantly evolved dual functions within one protein targeting the cell cycle machinery for viral replication.
doi:10.1371/journal.ppat.1003825
PMCID: PMC3873445  PMID: 24385906
9.  Cyclin D1 expression in transitional cell carcinoma of the bladder: correlation with p53, waf1, pRb and Ki67 
British Journal of Cancer  2001;84(2):270-275.
Normal cell proliferation is closely regulated by proteins called cyclins. One of these, cyclin D1, in combination with its corresponding cyclin-dependent kinase (cdk), is essential for G1/S phase transition. Cyclin/cdk complexes are generally inhibited by cyclin-dependent kinase inhibitors(ckis), some of which are induced by wild-type p53. The aims of this study were: to investigate levels of cyclin D1 expression in transitional cell carcinoma (TCC) of the bladder; to correlate these results with data concerning the expression of p53, waf1, pRb and Ki67; and to determine whether cyclin D1 expression could predict clinical outcome. Paraffin-sections from 150 newly diagnosed bladder tumours (Ta/T1 = 97; T2–T4 = 53) were stained for cyclin D1 using immunohistochemistry and a cyclin D1 index assigned. These results were correlated with data relating to the expression of p53 and waf1 by the same tumours. A representative subset of 54 tumours (Ta/T1 = 28; T2–T4 = 26) was also stained for Ki67 and 55 were stained for pRb. The clinical course of each patient was recorded and multivariate analyses of risk factors for tumour recurrence, stage progression and overall survival were performed. Positive staining for cyclin D1 was found in 83% of tumours. The staining pattern varied between tumours with nuclear, cytoplasmic or a combination of the two evident in different tumours. 89% of Ta/T1 and 74% of T2–T4 tumours showed nuclear staining with or without cytoplasmic staining. The median value for cyclin D1 staining was significantly higher in Ta/T1 tumours (41%) compared with T2–T4 tumours (8%, P< 0.005) with 26% of muscle-invasive tumours demonstrating absent staining. In addition, the median value for cyclin D1 staining was significantly higher in G1/G2 tumours (43%) compared with G3 tumours (14%, P< 0.005). There was a significant positive correlation between expression of cyclin D1 and waf1 expression (P< 0.0001) as well as pRb expression but not between cyclin D1 expression and expression of p53. Ki67 expression was significantly associated with increasing tumour stage (P< 0.005) and histological grade (P< 0.05) but did not correlate with cyclin D1 expression. A cyclin D1 index ≥ 8% was associated with significantly better survival in those patients with muscle-invasive disease (T2–T4). In addition, there was a significantly higher progression rate for those patients with Ta/T1 disease whose tumours demonstrated cytoplasmic cyclin D1 staining. These results indicate that cyclin D1 expression is significantly higher in low-stage, well differentiated bladder tumours and strongly correlates with waf1 expression. In a multivariate analysis, cyclin D1 expression is an independent prognostic indicator of survival in those patients with muscle-invasive disease. © 2001 Cancer Research Campaign http://www.bjcancer.com
doi:10.1054/bjoc.2000.1557
PMCID: PMC2363716  PMID: 11161387
cyclin D1; bladder; carcinoma; immunohistochemistry; survival
10.  Low molecular weight cyclin E is associated with p27-resistant, high-grade, high-stage and invasive bladder cancer 
Cell Cycle  2012;11(7):1468-1476.
Expression of low molecular weight (LMW) isoforms of cyclin E is a strong predictor of poor outcome in patients with breast cancer. The purpose of this study was to examine the expression of full-length and LMW cyclin E in bladder cancer cell lines and patient tumors. We used western blotting, immunoprecipitation and kinase assays to examine the expression and activity of key cell cycle-regulatory proteins in various human bladder cell lines, both tumorigenic and non-tumorigenic. We also analyzed cyclin E expression, kinase activity and immune complex binding partners in 43 tissue samples from grade 2 and 3 transitional cell carcinomas. Cyclin E was overexpressed and LMW isoforms were present only in bladder cancer cells. Overexpression of LMW isoforms of cyclin E and increased cyclin E kinase activity were both significantly associated with tumorigenicity of the bladder cell lines (p = 0.005 and 0.022, respectively). Binding of the cyclin-dependent kinase inhibitors p21 and p27 to LMW cyclin E did not inhibit the kinase activity of cyclin E and cyclin-dependent kinase 2 in primary tumor samples overexpressing LMW cyclin E. Full-length and LMW cyclin E were significantly overexpressed in grade 3 tumors compared with grade 2 tumors (p = 0.004). Finally, LMW cyclin E levels were significantly associated with a non-papillary growth pattern (p = 0.031) and invasiveness (p = 0.021) of the bladder tumors and poor overall survival (p = 0.06). These results suggest that LMW cyclin E can be used as a new prognostic marker for bladder cancer.
doi:10.4161/cc.19882
PMCID: PMC3350882  PMID: 22441703
cyclin E; p27; Cdk2 kinase; bladder cancer; cell cycle
11.  A Mechanism for the Inhibition of Neural Progenitor Cell Proliferation by Cocaine 
PLoS Medicine  2008;5(6):e117.
Background
Prenatal exposure of the developing brain to cocaine causes morphological and behavioral abnormalities. Recent studies indicate that cocaine-induced proliferation inhibition and/or apoptosis in neural progenitor cells may play a pivotal role in causing these abnormalities. To understand the molecular mechanism through which cocaine inhibits cell proliferation in neural progenitors, we sought to identify the molecules that are responsible for mediating the effect of cocaine on cell cycle regulation.
Methods and Findings
Microarray analysis followed by quantitative real-time reverse transcription PCR was used to screen cocaine-responsive and cell cycle-related genes in a neural progenitor cell line where cocaine exposure caused a robust anti-proliferative effect by interfering with the G1-to-S transition. Cyclin A2, among genes related to the G1-to-S cell cycle transition, was most strongly down-regulated by cocaine. Down-regulation of cyclin A was also found in cocaine-treated human primary neural and A2B5+ progenitor cells, as well as in rat fetal brains exposed to cocaine in utero. Reversing cyclin A down-regulation by gene transfer counteracted the proliferation inhibition caused by cocaine. Further, we found that cocaine-induced accumulation of reactive oxygen species, which involves N-oxidation of cocaine via cytochrome P450, promotes cyclin A down-regulation by causing an endoplasmic reticulum (ER) stress response, as indicated by increased phosphorylation of eIF2α and expression of ATF4. In the developing rat brain, the P450 inhibitor cimetidine counteracted cocaine-induced inhibition of neural progenitor cell proliferation as well as down-regulation of cyclin A.
Conclusions
Our results demonstrate that down-regulation of cyclin A underlies cocaine-induced proliferation inhibition in neural progenitors. The down-regulation of cyclin A is initiated by N-oxidative metabolism of cocaine and consequent ER stress. Inhibition of cocaine N-oxidative metabolism by P450 inhibitors may provide a preventive strategy for counteracting the adverse effects of cocaine on fetal brain development.
Investigating the mechanism of cocaine's effect on fetal brain development, Chun-Ting Lee and colleagues find that down-regulation of cyclin A by a cocaine metabolite inhibits neural proliferation.
Editors' Summary
Background.
Every year, cocaine abuse by mothers during pregnancy exposes thousands of unborn infants (fetuses) to this powerful and addictive stimulant. Maternal cocaine abuse during early pregnancy increases the risk of miscarriage; its use during late pregnancy slows the baby's growth and can trigger premature labor. Babies exposed to cocaine shortly before birth are often irritable and have disturbed sleep patterns. They can also be very sensitive to sound and touch and consequently hard to comfort. These problems usually resolve spontaneously within the first few weeks of life but some permanent birth defects are also associated with frequent cocaine abuse during pregnancy. In particular, babies exposed to cocaine before birth sometimes have small heads—an abnormality that generally indicates a small brain—and, although they usually have normal intelligence, the development of their thinking skills and language is often delayed, and they can have behavioral problems.
Why Was This Study Done?
Exposure to cocaine before birth clearly interferes with some aspects of brain development. More specifically, it reduces the number and position of neurons (the cells that transmit information in the form of electrical impulses around the body) within the brain. All neurons develop from neural progenitor cells, and previous research suggests that cocaine exposure before birth inhibits the proliferation of these cells in the developing brain. It would be useful to understand exactly how cocaine affects neural progenitor cells, because it might then be possible to prevent the drug's adverse effects on brain development. In this study, therefore, the researchers investigate the molecular mechanism that underlies cocaine's effect on neural progenitor cells.
What Did the Researchers Do and Find?
When the researchers investigated the effects of cocaine on AF5 cells (rat neural progenitor cells that grow indefinitely in the laboratory), they found that concentrations of cocaine similar to those measured in fetal brains after maternal drug exposure inhibited the proliferation of AF5 cells by blocking the “G1-to-S transition.” This is a stage that cells have to pass through between each round of cell division (the production of two daughter cells from one parent cell). Next, the researchers showed that cocaine-treated AF5 cells made much less cyclin A2, a protein that controls the G1-to-S transition, than untreated cells. Cocaine also decreased cyclin A2 levels in neural progenitor cells freshly isolated from human fetal brains and in fetal rat brains exposed to the drug while in their mother's womb. Treatment of AF5 cells with a cyclin A2 expression vector (a piece of DNA that directs the production of cyclin A2) counteracted the down-regulation of cyclin A2 and restored AF5 proliferation in the presence of cocaine. Other experiments indicate that the reduction of cyclin A2 by cocaine in AF5 cells involves the accumulation of “reactive oxygen species,” by-products of the breakdown of cocaine by a protein that is a member of a family of proteins called cytochrome P450. Finally, treatment of pregnant rats with cimetidine (which inhibits the action of cytochrome P450) counteracted both the inhibition of neural progenitor cell proliferation and the cyclin A2 down-regulation that cocaine exposure induced in the brains of their unborn pups.
What Do These Findings Mean?
These findings show that the cocaine-induced inhibition of neural progenitor cell proliferation involves, at least in part, interfering with the production (that is, causing down-regulation) of cyclin A2. They also show that this down-regulation is induced by the breakdown of cocaine by cytochrome P450, and that in both a rat cell line and in fetal rats, the cytochrome P450 inhibitor cimetidine (a drug that is already used clinically for stomach problems) can block the adverse effects of cocaine on the proliferation of neural progenitor cells. These findings need to be confirmed in animals more closely related to people than rats, and the long-term effects of cimetidine need to be investigated, in particular its effects on cocaine toxicity. Nevertheless these results raise the possibility that giving cimetidine or other drugs with similar effects to pregnant women who are addicted to cocaine might prevent some of the harm that their drug habit does to their unborn children, although it is not clear whether there is a dosage of cimetidine that might be both safe and adequate for this purpose.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050117.
A PLoS Medicine Perspective article by Steven Hyman further discusses this study
The US National Institute on Drug Abuse provides a fact sheet on cocaine (in English and Spanish)
The UK charity Release provides information and advice to the public and professionals about the law and drugs, including information about cocaine
MedlinePlus also provides a list of links to information about cocaine (in English and Spanish)
The March of Dimes Foundation, a US nonprofit organization for the improvement of child health, provides information about illicit drug use during pregnancy (in English and Spanish)
The Organization of Teratology Information Specialists also provides a fact sheet on cocaine and pregnancy (in English, Spanish, and French)
doi:10.1371/journal.pmed.0050117
PMCID: PMC2504032  PMID: 18593214
12.  The prognostic significance and value of cyclin D1, CDK4 and p16 in human breast cancer 
Introduction
Loss of the retinoblastoma protein tumor suppressor gene (RB) coding for a nuclear phosphoprotein that regulates the cell cycle is found in many human cancers and probably leads to disruption of the p16-cyclin D1-CDK4/6-RB pathway. Cyclin D1 is known to activate CDK4, which then phosphorylates the RB protein, leading to cell cycle progression. p16 inhibits CDK4, keeping RB hypophosphorylated and preventing cell cycle progression. The significance of these three markers, cyclin D1, CDK4 and p16, for breast cancer and carcinogenesis is nevertheless still controversial.
Methods
The material consisted of 102 formalin-fixed human breast cancer samples, in which cyclin D1, CDK4 and p16 expression was evaluated immunohistochemically. The amounts of cyclin D1 mRNA present were analyzed by quantitative real time PCR.
Results
High cyclin D1 expression statistically significantly correlated with lower tumor grade, estrogen and progesterone receptor positivity and lower proliferation activity in breast tumors and increased breast cancer-specific survival and overall survival. Tumors with high cyclin D1 protein had 1.8 times higher expression of cyclin D1 mRNA. CDK4 expression did not correlate with cyclin D1 expression or the survival data. p16 expression was associated with Human Epidermal Growth Factor Receptor 2 (HER2) negativity and increased breast cancer-specific survival and disease-free survival. No statistical correlations between cyclin D1, CDK4 and p16 were found.
Conclusions
Cyclin D1 was associated with a good breast cancer prognosis but functioned independently of CDK4. High cyclin D1 expression may be partially due to increased CCND1 transcription. p16 correlated with a better prognosis and may function without CDK4. In conclusion, it appears that cyclin D1, CDK4 and p16 function independently in human breast cancer.
doi:10.1186/bcr3376
PMCID: PMC3672746  PMID: 23336272
13.  Transgenic mice with mammary gland targeted expression of human cortactin do not develop (pre-malignant) breast tumors: studies in MMTV-cortactin and MMTV-cortactin/-cyclin D1 bitransgenic mice 
BMC Cancer  2006;6:58.
Background
In human breast cancers, amplification of chromosome 11q13 correlates with lymph node metastasis and increased mortality. To date, two genes located within this amplicon, CCND1 and EMS1, were considered to act as oncogenes, because overexpression of both proteins, respectively cyclin D1 and cortactin, correlated well with 11q13 amplification. Cyclin D1 is involved in cell cycle regulation and the F-actin-binding protein cortactin in cytoskeletal dynamics and cell migration. To study the role of cortactin in mammary gland tumorigenesis, we examined mouse mammary tumor virus (MMTV)-cortactin transgenic mice and MMTV-cortactin/-MMTV-cyclin D1 bitransgenic mice.
Methods
MMTV-cortactin transgenic mice were generated and intercrossed with previously described MMTV-cyclin D1 transgenic mice. Immunohistochemical, Northern and Western blot analyses were performed to study the expression of human transgene cortactin during mammary gland development and in mammary tumors. For tumor incidence studies, forced-bred, multiparous mice were used to enhance transgene expression in the mammary gland. Microscopical examination was performed using haematoxylin and eosin staining.
Results
Mammary gland tumors arose stochastically (incidence 21%) with a mean age of onset at 100 weeks. This incidence, however, did not exceed that of aged-matched control FVB/N mice (38%), which unexpectedly, also developed spontaneous mammary gland tumors. We mimicked 11q13 amplification by generating MMTV-cortactin/-MMTV-cyclin D1 bitransgenic mice but did not observe any synergistic effect of cortactin on cyclin D1-induced mammary hyperplasias or carcinomas, nor development of distant metastasis.
Conclusion
From this study, we conclude that development of (pre-malignant) breast tumors in either wild type or MMTV-cyclin D1 mice was not augmented due to mammary gland targeted overexpression of human cortactin.
doi:10.1186/1471-2407-6-58
PMCID: PMC1450299  PMID: 16536875
14.  Targeting cyclin B1 inhibits proliferation and sensitizes breast cancer cells to taxol 
BMC Cancer  2008;8:391.
Background
Cyclin B1, the regulatory subunit of cyclin-dependent kinase 1 (Cdk1), is essential for the transition from G2 phase to mitosis. Cyclin B1 is very often found to be overexpressed in primary breast and cervical cancer cells as well as in cancer cell lines. Its expression is correlated with the malignancy of gynecological cancers.
Methods
In order to explore cyclin B1 as a potential target for gynecological cancer therapy, we studied the effect of small interfering RNA (siRNA) on different gynecological cancer cell lines by monitoring their proliferation rate, cell cycle profile, protein expression and activity, apoptosis induction and colony formation. Tumor formation in vivo was examined using mouse xenograft models.
Results
Downregulation of cyclin B1 inhibited proliferation of several breast and cervical cancer cell lines including MCF-7, BT-474, SK-BR-3, MDA-MB-231 and HeLa. After combining cyclin B1 siRNA with taxol, we observed an increased apoptotic rate accompanied by an enhanced antiproliferative effect in breast cancer cells. Furthermore, control HeLa cells were progressively growing, whereas the tumor growth of HeLa cells pre-treated with cyclin B1 siRNA was strongly inhibited in nude mice, indicating that cyclin B1 is indispensable for tumor growth in vivo.
Conclusion
Our data support the notion of cyclin B1 being essential for survival and proliferation of gynecological cancer cells. Concordantly, knockdown of cyclin B1 inhibits proliferation in vitro as well as in vivo. Moreover, targeting cyclin B1 sensitizes breast cancer cells to taxol, suggesting that specific cyclin B1 targeting is an attractive strategy for the combination with conventionally used agents in gynecological cancer therapy.
doi:10.1186/1471-2407-8-391
PMCID: PMC2639606  PMID: 19113992
15.  Cyclin D3 compensates for the loss of Cyclin D1 during ErbB2-induced mammary tumor initiation and progression 
Cancer research  2011;71(24):7513-7524.
Cyclin D1 regulates cell proliferation and is a candidate molecular target for breast cancer therapy. The current work addresses whether Cyclin D1 is indispensable for ErbB2-associated mammary tumor initiation and progression using a breast cancer model in which this cell cycle regulator can be genetically ablated prior to or after neoplastic transformation. Deficiency in Cyclin D1 delayed tumor onset but did not prevent the occurrence of mammary cancer in mice overexpressing wildtype ErbB2. The lack of Cyclin D1 was associated with a compensatory upregulation of Cyclin D3, which explains why the targeted downregulation of Cyclin D1 in established mammary tumors had no effect on cancer cell proliferation. Cyclin D1 and D3 are overexpressed in human breast cancer cell lines and primary invasive breast cancers, and Cyclin D3 frequently exceeded the expression of Cyclin D1 in ErbB2-positive cases. The simultaneous inhibition of both cyclins in mammary tumor cells reduced cancer cell proliferation in vitro and decreased the tumor burden in vivo. Collectively, the results of this study suggest that only the combined inhibition of Cyclin D1 and D3 might be a suitable strategy for breast cancer prevention and therapy.
doi:10.1158/0008-5472.CAN-11-1783
PMCID: PMC3242818  PMID: 22037875
Cyclin D; Gene Targeting; Tetracycline Transactivator; ErbB2; Mammary Gland Development; Breast Cancer
16.  Role of Cyclin D1 as a Mediator of c-Met and β-Catenin Induced Hepatocarcinogenesis 
Cancer research  2009;69(1):253-261.
Activation of c-Met signaling and β-catenin mutations are frequent genetic events observed in liver cancer development. Recently, we demonstrated that activated β-catenin can cooperate with c-Met to induce liver cancer formation in a mouse model. Cyclin D1 is an important cell cycle regulator that is considered to be a downstream target of β-catenin. To determine the importance of cyclin D1 as a mediator of c-Met and β-catenin induced hepatocarcinogenesis, we investigated the genetic interactions between cyclin D1, β-catenin and c-Met in liver cancer development using mouse models. We co-expressed cyclin D1 with c-Met in mice and found cyclin D1 to cooperate with c-Met to promote liver cancer formation. Tumors induced by cyclin D1/c-Met had a longer latency period, formed at a lower frequency, and appeared to be more benign compared to those induced by β-catenin/c-Met. In addition, when activated β-catenin and c-Met were co-injected into cyclin D1 null mice, liver tumors developed despite the absence of cyclin D1. Intriguingly, we observed a moderate accelerated tumor growth and increased tumor malignancy in these cyclin D1 null mice. Molecular analysis demonstrated an up-regulation of cyclin D2 expression in cyclin D1 null tumor samples, indicating that cyclin D2 may replace cyclin D1 in hepatic tumorigenesis. Together, our results suggest that cyclin D1 functions as a mediator of β-catenin during HCC pathogenesis, although other molecules may be required to fully propagate β-catenin signaling. Moreover, our data suggest that cyclin D1 expression is not essential for liver tumor development induced by c-met and β-catenin.
doi:10.1158/0008-5472.CAN-08-2514
PMCID: PMC2628201  PMID: 19118010
HCC; Cyclin D1; Wnt pathway; β-catenin; c-Met
17.  Expression of molecular biomarkers in primary breast tumors implanted into a surrogate host: increased levels of cyclins correlate with tumor progression. 
Molecular Medicine  1997;3(4):273-283.
BACKGROUND: The overexpression or amplification of tumor suppressor and proto-oncogenes are important factors in the progression of breast cancer. Recent attention has focused on the cyclin genes, whose involvement in signal transduction pathways regulate cell cycle progression. The amplification of the cyclins D1 and D3 genes usually leads to loss of normal growth control and is thought to play an important growth regulatory role in tumor development and progression. In this report, we investigate the association of altered cyclin expression with other prognostic indicators (histological grade, lymph node status, estrogen receptor, p53, and c-erbB-2) in the progression of human breast cancer. MATERIALS AND METHODS: Surgical tumor specimens were obtained from 16 breast tubular ductal, and invasive ductal carcinomas and grafted onto gnotobiotic nude (nu/nu) mice. The expression diversity and distribution of the localization of the protein products of the c-erbB-2, cyclins D1 and D3, p53, and estrogen receptor were characterized immunohistochemically and the results in the original tumor (T0) were compared with those in the tumors that developed in nude mice (T1) xenografts. RESULTS: The T0 tumors exhibited a diversity of cellular morphology in the tumor matrix and diversity in expression of these proteins. These specific changes were also preserved in the T1 tumors. Whereas 67% of the T1 tumors exhibited high numbers of estrogen receptorpositive nuclei, only 50% of these tumors grew when grafted onto nude mice. The histological grade (14/15 were G2 to G3) and metastatic malignancy in the lymph nodes (10/15) did not appear to be related to tumor growth in the nude mouse. There was no relationship between those tumors which exhibited high percentages of c-erbB-2- and p53-positive cells and growth in nude mice. A strong association (p < 0.001) was observed between the overexpression of cyclin D1 transcripts in the T0 tumors and the continued growth of the T1 tumors in nude mice. In the T1 tumors, both cyclins D1 and D3, estrogen receptor, and p53 were observed in 49% to 86% of the cells of the T1 tumors examined; the number of cells expressing c-erbB-2 protein varied widely in these tumors. CONCLUSIONS: The results indicate that the tumor matrix exhibits a diversity in the level of phenotypic expression of genes involved in cellular growth of breast tumors in both the T0 or T1 host environment. Changes in cyclin activity appear to correlate with the vigorous level of breast tumor growth and progression.
Images
PMCID: PMC2230072  PMID: 9131589
18.  Longer Survival in Patients with Breast Cancer with Cyclin D1 Over-Expression after Tumor Recurrence: Longer, but Occupied with Disease 
Journal of Breast Cancer  2014;17(1):47-53.
Purpose
The effect of cyclin D1 overexpression on breast cancer outcomes and prognosis is controversial, even though amplification of the cyclin D1 gene, CCND1, has been shown to be associated with early relapse and poor prognosis. In this study, we examined the relationship between cyclin D1 overexpression and disease-specific survival (DSS). We also analyzed survival in patients who experienced recurrence.
Methods
We retrospectively analyzed data from patients diagnosed with ductal carcinoma between April 2005 and December 2010. We examined clinicopathologic factors associated with cyclin D1 overexpression and analyzed the influence of cyclin D1 on recurrence-free survival and DSS.
Results
We identified 236 patients diagnosed with primary breast cancer who completed all phases of their primary treatment. Cyclin D1 overexpression was significantly associated with longer DSS (5-year DSS, 89.9% in patients without cyclin D1 overexpression vs. 98.9% in patients with cyclin D1 overexpression; p=0.008). Multivariate analysis also found that patients with cyclin D1 overexpressing tumors had significantly longer disease-specific survival than patients whose tumors did not overexpress cyclin D1, with a hazard ratio for disease-specific mortality of 7.97 (1.17-54.22, p=0.034). However, in the group of patients who experienced recurrence, cyclin D1 overexpression was not significantly associated with recurrence-free survival. Cyclin D1 overexpression was significantly associated with increased survival after disease recurrence, indicating that cyclin D1 overexpression might be indicative of more indolent disease progression after metastasis.
Conclusion
Cyclin D1 overexpression is associated with longer DSS, but not recurrence-free survival, in patients with breast cancer. Longer postrecurrence survival could explain the apparent inconsistency between DSS and recurrence-free survival. Patients with cyclin D1-overexpressing tumors survive longer, but with metastatic disease after recurrence. This information should spark the urgent development of tailored therapies to cure these patients.
doi:10.4048/jbc.2014.17.1.47
PMCID: PMC3988342  PMID: 24744797
Breast neoplasms; Cyclin D1; Disease-specific survival; Recurrence
19.  Zinc Fingers and Homeoboxes 2 Inhibits Hepatocellular Carcinoma Cell Proliferation and Represses Expression of Cyclins A and E 
Gastroenterology  2012;142(7):1559-1570.e2.
BACKGROUND & AIMS
Zinc-fingers and homeoboxes 2 (ZHX2) represses transcription of several genes associated with liver cancer. However, little is known about the role of ZHX2 in development of hepatocellular carcinoma (HCC). We investigated the mechanisms by which ZHX2 might affect proliferation of HCC cells.
METHODS
We overexpressed and knocked down ZHX2 in HCC cells and analyzed the effects on proliferation, colony formation, and the cell cycle. We also analyzed the effects of ZHX2 overexpression in growth of HepG2.2.15 tumor xenografts in nude mice. Chromatin immunoprecipitation and luciferase reporter assays were used to measure binding of ZHX2 target promoters. Levels of ZHX2 in HCC samples were evaluated by immunohistochemistry.
RESULTS
ZHX2 overexpression significantly reduced proliferation of HCC cells and growth of tumor xenografts in mice; it led to G1 arrest and reduced levels of cyclins A and E in HCC cell lines. ZHX2 bound to promoter regions of CCNA2 (which encodes Cyclin A) and CCNE1 (which encodes cyclin E) and inhibited their transcription. Knockdown of cyclin A or cyclin E reduced the increased proliferation mediated by ZHX2 knockdown. Nuclear localization of ZHX2 was required for it to inhibit proliferation of HCC cells in culture and in mice. Nuclear localization of ZHX2 was reduced in human HCC samples, even in small tumors (diameter<5 cm), compared to adjacent non-tumor tissues. Moreover, reduced nuclear levels of ZHX2 correlated with reduced survival times of patients, high levels of tumor microvascularization, and hepatocyte proliferation.
CONCLUSIONS
ZHX2 inhibits HCC cell proliferation, by preventing expression of cyclins A and E, and reduces growth of xenograft tumors in mice. Loss of nuclear ZHX2 might be an early step in the development of HCC.
doi:10.1053/j.gastro.2012.02.049
PMCID: PMC3367107  PMID: 22406477
mouse model; carcinogenesis; shRNA; CCK-8
20.  Human cyclin E, a nuclear protein essential for the G1-to-S phase transition. 
Molecular and Cellular Biology  1995;15(5):2612-2624.
Cyclin E was first identified by screening human cDNA libraries for genes that would complement G1 cyclin mutations in Saccharomyces cerevisiae and has subsequently been found to have specific biochemical and physiological properties that are consistent with it performing a G1 function in mammalian cells. Most significantly, the cyclin E-Cdk2 complex is maximally active at the G1/S transition, and overexpression of cyclin E decreases the time it takes the cell to complete G1 and enter S phase. We have now found that mammalian cells express two forms of cyclin E protein which differ from each other by the presence or absence of a 15-amino-acid amino-terminal domain. These proteins are encoded by alternatively spliced mRNAs and are localized to the nucleus during late G1 and early S phase. Fibroblasts engineered to constitutively overexpress either form of cyclin E showed elevated cyclin E-dependent kinase activity and a shortened G1 phase of the cell cycle. The overexpressed cyclin E protein was detected in the nucleus during all cell cycle phases, including G0. Although the cyclin E protein could be overexpressed in quiescent cells, the cyclin E-Cdk2 complex was inactive. It was not activated until 6 to 8 h after readdition of serum, 4 h earlier than the endogenous cyclin E-Cdk2. This premature activation of cyclin E-Cdk2 was consistent with the extent of G1 shortening caused by cyclin E overexpression. Microinjection of affinity-purified anti-cyclin E antibodies during G1 inhibited entry into S phase, whereas microinjection performed near the G1/S transition was ineffective. These results demonstrate that cyclin E is necessary for entry into S phase. Moreover, we found that cyclin E, in contrast to cyclin D1, was required for the G1/S transition even in cells lacking retinoblastoma protein function. Therefore, cyclins E and D1 control two different transitions within the human cell cycle.
PMCID: PMC230491  PMID: 7739542
21.  LMW-E/CDK2 Deregulates Acinar Morphogenesis, Induces Tumorigenesis, and Associates with the Activated b-Raf-ERK1/2-mTOR Pathway in Breast Cancer Patients 
PLoS Genetics  2012;8(3):e1002538.
Elastase-mediated cleavage of cyclin E generates low molecular weight cyclin E (LMW-E) isoforms exhibiting enhanced CDK2–associated kinase activity and resistance to inhibition by CDK inhibitors p21 and p27. Approximately 27% of breast cancers express high LMW-E protein levels, which significantly correlates with poor survival. The objective of this study was to identify the signaling pathway(s) deregulated by LMW-E expression in breast cancer patients and to identify pharmaceutical agents to effectively target this pathway. Ectopic LMW-E expression in nontumorigenic human mammary epithelial cells (hMECs) was sufficient to generate xenografts with greater tumorigenic potential than full-length cyclin E, and the tumorigenicity was augmented by in vivo passaging. However, cyclin E mutants unable to interact with CDK2 protected hMECs from tumor development. When hMECs were cultured on Matrigel, LMW-E mediated aberrant acinar morphogenesis, including enlargement of acinar structures and formation of multi-acinar complexes, as denoted by reduced BIM and elevated Ki67 expression. Similarly, inducible expression of LMW-E in transgenic mice generated hyper-proliferative terminal end buds resulting in enhanced mammary tumor development. Reverse-phase protein array assay of 276 breast tumor patient samples and cells cultured on monolayer and in three-dimensional Matrigel demonstrated that, in terms of protein expression profile, hMECs cultured in Matrigel more closely resembled patient tissues than did cells cultured on monolayer. Additionally, the b-Raf-ERK1/2-mTOR pathway was activated in LMW-E–expressing patient samples, and activation of this pathway was associated with poor disease-specific survival. Combination treatment using roscovitine (CDK inhibitor) plus either rapamycin (mTOR inhibitor) or sorafenib (a pan kinase inhibitor targeting b-Raf) effectively prevented aberrant acinar formation in LMW-E–expressing cells by inducing G1/S cell cycle arrest. LMW-E requires CDK2–associated kinase activity to induce mammary tumor formation by disrupting acinar development. The b-Raf-ERK1/2-mTOR signaling pathway is aberrantly activated in breast cancer and can be suppressed by combination treatment with roscovitine plus either rapamycin or sorafenib.
Author Summary
Effective cancer treatment should include targeting not only drivers of tumorigenicity but also the downstream signaling pathways that these drivers activate. Special attention has to be given to the model systems that identify these targets and interrogating if these targets are poor prognostic indicators in patients. Using cell lines cultured on plastic and extracellular matrix (Matrigel) and comparing their proteomic profiles to breast cancer tumor samples, we demonstrated that overexpression of LMW-E is concomitant with activation of the b-Raf-ERK1/2-mTOR pathway. Using mouse models, we show that induction of LMW-E is sufficient to induce mammary tumor development in vivo. Next, cells established from the tumors were treated with combination therapy targeting the LMW-E/CDK2 complex and the b-Raf-ERK1/2-mTOR pathway. Results revealed that this combination therapy effectively inhibited the altered proliferation of these cells. Most significantly, we showed that breast cancer patients whose tumors overexpress both LMW-E and different components of the b-Raf-ERK1/2-mTOR pathway have the worst prognosis. In summary, through the use of multiple in vitro and in vivo model systems and translating the findings to clinical specimens, we have identified a novel targeted therapy in breast cancer patients whose tumors overexpress LMW-E.
doi:10.1371/journal.pgen.1002538
PMCID: PMC3315462  PMID: 22479189
22.  Clinico-prognostic value of D-type cyclins and p27 in laryngeal cancer patients: a review 
Summary
Despite recent improvements in surgical and radiation therapy, failures still occur in patients with laryngeal squamous cell carcinomas, which may have a very different clinical outcome even when their clinical and histopathological characteristics are similar. The apparent inadequacy of “traditional” prognostic factors in predicting the clinical evolution of laryngeal squamous cell carcinomas has led to attempts to develop additional markers capable of distinguishing patients with a good prognosis from those who are more likely to relapse. A number of studies have demonstrated a relationship between tumourigenesis and alterations in the expression of cyclins, cyclin-dependent kinases and cyclin-dependent kinase inhibitors, but the data regarding laryngeal squamous cell carcinomas are somewhat conflicting. Herein a review is made of the published literature concerning the clinico-prognostic role of cyclin D1, D3 and p27, and personal data are described concerning laryngeal squamous cell carcinoma patients who underwent surgical resection at the ENT Department of the University of Milan. The results of our multivariate analyses demonstrated that cyclin D1, p27 and cyclin D3 overexpression are statistically significant predictors of disease-free survival (p = 0.0238, p = 0.0001 and p = 0.0217, respectively); the statistical correlation with overall survival was significant in the case of p27 (p = 0.0009) and cyclin D3 (p = 0.0189), and borderline in the case of cyclin D1 (p = 0.0622). In relation to cyclin D1/p27 coexpression, the patients with a cyclin D1-/p27+ phenotype showed the best prognosis, those with a cyclin D1+/p27+ or cyclin D1-/p27- phenotype, an intermediate prognosis, and those with a cyclin D1+/p27- phenotype, the poorest prognosis (p = 0.0001 and p = 0.0001 for trend for disease-free survival; p = 0.0015 and p = 0.0008 for trend for overall survival). In the case of cyclin D1/cyclin D3 coexpression, the patients with cyclin D1+/cyclin D3+ tumours had the poorest overall survival, those with cyclin D1-/cyclin D3+ or cyclin D1+/cyclin D3- tumours showed intermediate course, and those with cyclin D1-/cyclin D3- tumours had the most favourable outcome (p = 0.0002). The findings of this review indicate that both types of cyclin D and p27 are involved in the genesis of laryngeal squamous cell carcinomas, and that immunohistochemical evaluations of biopsy samples may provide useful additional markers capable of identifying subgroups of patients with a poor prognosis who can be treated by means of more aggressive surgery, adjuvant radiotherapy and chemotherapy, as well as those requiring a closer and more prolonged follow-up. Finally, preliminary results suggest that the administration of new molecular therapies that exert their antitumoural activities by functionally subverting the pathways regulated by D-type cyclins and their cyclin-dependent kinase counterparts may represent a further therapeutic modality for patients with refractory head and neck squamous cell carcinomas.
PMCID: PMC2639874  PMID: 16116829
Larynx; Carcinoma; Prognosis; Cyclin D1; Cyclin D3; p27
23.  ErbB2 Induces Notch1 Activity and Function in Breast Cancer Cells 
The ErbB2 (Her2/neu epidermal growth receptor family) oncogene is overexpressed in 30% to 40% of human breast cancers. Cyclin D1 is the regulatory subunit of the holoenzyme that phosphorylates and inactivates the retinoblastoma (pRb) tumor suppressor and is an essential downstream target of ErbB2-induced tumor growth. Herein, we demonstrate that ErbB2 induces the activity of the Notch signaling pathway. ErbB2 induction of DNA synthesis, contact-independent growth, and mammosphere induction required Notch1. ErbB2-induced cyclin D1 and cyclin D1 expression was sufficient to induce Notch1 activity, and conversely, genetic deletion of Notch1 in mammary epithelial cells using floxed Notch (Notchfl/fl ) mice demonstrated that cyclin D1 is induced by Notch1. Genetic deletion of cyclin D1 or small interfering RNA (siRNA) to cyclin D1-reduced Notch1 activity and reintroduction of cyclin D1 into cyclin D1-deficient cells restored Notch1 activity through the inhibition of Numb, an endogenous inhibitor of Notch1 activity. Thus, cyclin D1 functions downstream as a genetic target of Notch1, amplifies Notch1 activity by repressing Numb, and identifies a novel pathway by which ErbB2 induces Notch1 activity via the induction of cyclin D1.
doi:10.1111/j.1752-8062.2008.00041.x
PMCID: PMC3590841  PMID: 20443831
cancer biology; oncogenes; signal transduction
24.  Downregulation of the tumor-suppressor miR-16 via progestin-mediated oncogenic signaling contributes to breast cancer development 
Introduction
Experimental and clinical evidence points to a critical role of progesterone and the nuclear progesterone receptor (PR) in controlling mammary gland tumorigenesis. However, the molecular mechanisms of progesterone action in breast cancer still remain elusive. On the other hand, micro RNAs (miRNAs) are short ribonucleic acids which have also been found to play a pivotal role in cancer pathogenesis. The role of miRNA in progestin-induced breast cancer is poorly explored. In this study we explored progestin modulation of miRNA expression in mammary tumorigenesis.
Methods
We performed a genome-wide study to explore progestin-mediated regulation of miRNA expression in breast cancer. miR-16 expression was studied by RT-qPCR in cancer cell lines with silenced PR, signal transducer and activator of transcription 3 (Stat3) or c-Myc, treated or not with progestins. Breast cancer cells were transfected with the precursor of miR-16 and proliferation assays, Western blots or in vivo experiments were performed. Target genes of miR-16 were searched through a bioinformatical approach, and the study was focused on cyclin E. Reporter gene assays were performed to confirm that cyclin E 3'UTR is a direct target of miR-16.
Results
We found that nine miRNAs were upregulated and seven were downregulated by progestin in mammary tumor cells. miR-16, whose function as a tumor suppressor in leukemia has already been shown, was identified as one of the downregulated miRNAs in murine and human breast cancer cells. Progestin induced a decrease in miR-16 levels via the classical PR and through a hierarchical interplay between Stat3 and the oncogenic transcription factor c-Myc. A search for miR-16 targets showed that the CCNE1 gene, encoding the cell cycle regulator cyclin E, contains conserved putative miR-16 target sites in its mRNA 3' UTR region. We found that, similar to the molecular mechanism underlying progestin-modulated miR-16 expression, Stat3 and c-Myc participated in the induction of cyclin E expression by progestin. Moreover, overexpression of miR-16 abrogated the ability of progestin to induce cyclin E upregulation, revealing that cyclin E is a novel target of miR-16 in breast cancer. Overexpression of miR-16 also inhibited progestin-induced breast tumor growth in vitro and in vivo, demonstrating for the first time, a role for miR-16 as a tumor suppressor in mammary tumorigenesis. We also found that the ErbB ligand heregulin (HRG) downregulated the expression of miR-16, which then participates in the proliferative activity of HRG in breast tumor cells.
Conclusions
In this study, we reveal the first progestin-regulated miRNA expression profile and identify a novel role for miR-16 as a tumor suppressor in progestin- and growth factor-induced growth in breast cancer.
doi:10.1186/bcr3187
PMCID: PMC3446340  PMID: 22583478
25.  Identification of kinetin riboside as a repressor of CCND1 and CCND2 with preclinical antimyeloma activity  
The Journal of Clinical Investigation  2008;118(5):1750-1764.
Knockout and transgenic studies in mice demonstrate that normal somatic tissues redundantly express 3 cyclin D proteins, whereas tumor cells seem dependent on a single overexpressed cyclin D. Thus, selective suppression of the individual cyclin D deregulated in a tumor represents a biologically valid approach to targeted cancer therapy. In multiple myeloma, overexpression of 1 of the cyclin D proteins is a ubiquitous feature, unifying at least 7 different initiating genetic events. We demonstrate here that RNAi of genes encoding cyclin D1 and cyclin D2 (CCND1 and CCND2, respectively) inhibits proliferation and is progressively cytotoxic in human myeloma cells. By screening a chemical library using a cell-based assay for inhibition of CCND2 trans-activation, we identified the plant cytokinin kinetin riboside as an inhibitor of CCND2 trans-activation. Kinetin riboside induced marked suppression of CCND2 transcription and rapidly suppressed cyclin D1 and D2 protein expression in primary myeloma cells and tumor lines, causing cell-cycle arrest, tumor cell–selective apoptosis, and inhibition of myeloma growth in xenografted mice. Mechanistically, kinetin riboside upregulated expression of transcription repressor isoforms of cAMP-response element modulator (CREM) and blocked both trans-activation of CCND2 by various myeloma oncogenes and cis-activation of translocated CCND1, suggesting induction of an overriding repressor activity that blocks multiple oncogenic pathways targeting cyclin D genes. These data support targeted repression of cyclin D genes as a therapeutic strategy for human malignancies.
doi:10.1172/JCI34149
PMCID: PMC2323188  PMID: 18431519

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