High Cyclin D1 Expression Research Articles

Intrinsic subtype and its clinical significance in early node-negative breast cancer: Results from two randomized trials of adjuvant chemoendocrine therapy.

504 Background: Gene expression profiling has identified four main intrinsic subtypes of breast cancer which have distinct clinical behaviours and responses to therapy. A simplified immunohistochemical (IHC) panel can provide meaningful discrimination and help guide patient management. Methods: Using tissue microarrays (TMA) created from two randomized trials of adjuvant chemoendocrine therapy in node negative early breast cancer (IBCSG VIII and IX,n= 1220) we assessed intrinsic subtype as follows: “luminal A” (LA): ER and/or PR present, Ki-67 low (<median: 19%), HER2-; “Luminal B” (LB): ER and/or PR present, Ki-67 high(>=19% median) and/or HER2+ (IHC 3+ or FISH amplified); HER2 enriched: ER and PR negative, HER2 positive (IHC 3+ or FISH amplified), Triple Negative (TNP): ER, PR and HER2 negative. p53+ and high cyclin D1 expression were then assessed to determine if they provided any further additive discriminatory value. Results: The standard definitions of intrinsic subtype demonstrated a significant difference in disease-free survival between the subtypes in both trials VIII and IX (p= 0.02), but this was not significant in multivariate analysis. The discrimination between luminal A and B tumors could not be improved upon by the addition of p53 or cyclin D1 expression. The standard definitions demonstrated that TNP and HER2-enriched tumors benefited from the addition of chemotherapy to endocrine therapy, but LA and LB did not. p53+ was associated with a poorer prognosis in ER+ luminal tumors but improved prognosis in ER- non-luminal tumors (Interaction p = 0.002). Conclusions: Intrinsic subtyping using a panel of ER, PR, HER2 and Ki67 provides meaningful prognostic information to help guide patient management in early breast cancer. Whilst it predicts benefit from the addition of chemotherapy in TNP and HER2-enriched tumors it did not in this series do so for LB. Although p53 status did not improve the definition of luminal tumors, p53+ appeared to have a divergent effect on outcome dependent on ER status.

1896 CYCLIN D1 EXPRESSION INDEPENDENTLY PREDICTS SURVIVAL AND RESPONSE TO CHEMOTHERAPY IN LYMPH NODE POSITIVE BLADDER CANCER

You have accessJournal of UrologyBladder Cancer: Metastatic Disease/Staging1 Apr 20121896 CYCLIN D1 EXPRESSION INDEPENDENTLY PREDICTS SURVIVAL AND RESPONSE TO CHEMOTHERAPY IN LYMPH NODE POSITIVE BLADDER CANCER Roland Seiler, Achim Fleischmann, Diana Rotzer, and George N. Thalmann Roland SeilerRoland Seiler Bern, Switzerland More articles by this author , Achim FleischmannAchim Fleischmann Bern, Switzerland More articles by this author , Diana RotzerDiana Rotzer Bern, Switzerland More articles by this author , and George N. ThalmannGeorge N. Thalmann Bern, Switzerland More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.2052AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Cyclin D1 (CD1) expression predicts survival and also response to chemotherapy in different cancers. The prognostic impact of CD1 expression in lymph node positive bladder cancer and its potential to predict chemosensibility has never been determined. METHODS Hundred and fifty two lymph node positive patients with urothelial bladder cancer underwent cystectomy and pelvic lymphadenectomy. A tissue microarray was constructed with two samples from the primary tumor and two from lymph node metastasis per patient. In each spot, percentage of stained cancer cells was determined. Expression data were correlated with tumor features and cancer-specific survival (CSS). RESULTS Nuclear and cytoplasmic CD1 expression were significantly (p<0.05) up-regulated in metastases (median nuclear/cytoplasmic expression %50%/15%) compared to primary tumors (median nuclear/cytoplasmic expression: 30%/0%). CD1 expression was not correlated with tumor features (TNM stage; number, total diameter and extranodal extension of metastases). High nuclear CD1 expression in metastases predicted early death significantly (p=0.001) and independently (p=0.01) from other risk factors. Subgroup analyses showed that survival stratification was best in patients without any chemotherapy, all patients with high nuclear Cyclin D1 expression died during the first 2.5 years. Importantly, survival of this high risk subgroup increased dramatically when any chemotherapy (5-year CSS: 22%) and particularly platin-based chemotherapy (5-year CSS: 37%) was applied while the low risk group did not profit substantially. CD1 in all other tumor cell compartments failed to stratify survival. CONCLUSIONS Nuclear CD1 expression in lymph node metastases of bladder cancer predicts cancer related death independently and is a strong predictor of favorable response to chemotherapy. This prognostic and predictive information may help to identify high-risk patients and personalize adjuvant therapies, thus improving patient management. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e765 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Roland Seiler Bern, Switzerland More articles by this author Achim Fleischmann Bern, Switzerland More articles by this author Diana Rotzer Bern, Switzerland More articles by this author George N. Thalmann Bern, Switzerland More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Cyclin D1 Induces Chromosomal Instability

We developed mouse model systems to investigate the potential for cyclin D1 to induce CIN in vivo. In a mammary gland specific Tet-inducible model the acute expression profile regulated by cyclin D1 after 7 days was enriched in genes that rank highly with CIN. We also used a mammary gland targeted model (MMTV) to continuously express cyclin D1. The mice started to develop mammary gland tumors at 400 days and the tumor-free incidence was 40% in MMTV-cyclin D1. The gene expression profile of the tumors showed enrichment for the CIN signature. We next compared cyclin D1 expression and the highest ranking CIN genes to a breast cancer expression database and discovered that expression of genes promoting CIN are highly enriched in luminal subtype and that high cyclin D1 and CIN expression correlate specifically in the luminal B subtype. There is increasing interest in employing drugs in the clinic that exploit CIN in tumors. The high CIN expression index in luminal B breast cancer provides a basis for using Cdk and CIN inhibitors as a targeted therapeutic approach.

EpCAM regulates cell cycle progression via control of cyclin D1 expression

The epithelial cell adhesion molecule (EpCAM) is an integral transmembrane protein that is frequently overexpressed in embryonic stem cells, tissue progenitors, carcinomas and cancer-initiating cells. In cancer cells, expression of EpCAM is associated with enhanced proliferation and upregulation of target genes including c-myc. However, the exact molecular mechanisms underlying the observed EpCAM-dependent cell proliferation remained unexplored. Here, we show that EpCAM directly affects cell cycle progression via its capacity to regulate the expression of cyclin D1 at the transcriptional level and depending on the direct interaction partner FHL2 (four-and-a-half LIM domains protein 2). As a result, downstream events such as phosphorylation of the retinoblastoma protein (Rb) and expression of cyclins E and A are similarly affected. In vivo, EpCAM expression strength and pattern are both positively correlated with the proliferation marker Ki67, high expression and nuclear localisation of cyclin D1, and Rb phosphorylation. Thus, EpCAM enhances cell cycle progression via the classical cyclin-regulated pathway.

P4-01-22: Cyclin D1 and Its Prognostic Value in Planning of Endocrine Therapy for Women of Postmenopausal Age with Breast Cancer.

Abstract Background: Nowadays tamoxifen still remains the primary drug in breast cancer endocrine therapy. However, its application is limited due to the resistance of tumor cells. The search of adequate biomarkers is one of the most actual problems in prognosis of effectiveness of tamoxifen adjuvant therapy. The most perspective biomarker is cell cycle regulator cyclin D. Besides its central role in cell cycle regulation cyclin D1 modulates the estrogen receptors activity and can influence on the effectiveness of treatment with antiestrogenes and aromatase inhibitors. The objective of our work was to evaluate the effectiveness of tamoxifen in adjuvant therapy of hormone-receptor-positive breast cancer in women of postmenopausal age with cyclin D1 expression. Material and Methods: To evaluate the effectiveness of tamoxifen adjuvant application in women with cyclin D1-positive T1-4N0-3M0 breast cancer we have researched 2 retrospective groups of 70 patients that have been on regular medical check-up for a period of 5 years or that had previously undergone treatment. On the basis of the archive histological material we have revealed cyclin D1 in tumor cells. Results: The expression of cyclin was classified in 4 levels: negative, low, moderate and high. Patients with lack of cyclin D1 expression or with low quantitative value (according to our data less than 30%) have no neoplastic process progression throughout the 5 years of tamoxifen adjuvant therapy. These women were both with axillary lymph nodes affection and without it (+N; -N). On the contrary, women with moderate and high cyclin D1 expression (more than 30%) had a relapse of tumor and distant metastasis is prognosed in 5 years of observation (p=0,005). Moderate level of cyclin D1 expression was observed in 45 (64%) patients and 28 (62%) of them had progression with metastasis in bones, 9 (20%) metastasis in soft tissues, 2 (4%) metastasis in lungs and 6 (14%) relapse in postoperative scar. High expression was revealed in 25 (35%) women and 17 (68 %) of them had bone affection, 7 (28%) soft tissue metastasis and 2 (4%) tumor relapse in postoperative scar. Lung metastasis were not observed. The average period of tumor relapse and progression of neoplastic process in patients with cyclin D1-positive breast cancer is 20 months. Discussion: The results of our work suggest that women with hormone-receptor-positive cyclin D1-negative breast cancer on early stages have more prolonged non-relapse period during the tamoxifen adjuvant therapy. Patients with cyclin D1-positive breast cancer are less sensitive to tamoxifen treatment and in adjuvant regime should receive therapy with other effective equivalent drugs (aromatase inhibitors). Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-01-22.

Differential expression of cyclin D1 in human pituitary tumors: relation to MIB-1 and p27/Kip1 labeling indices

BackgroundPituitary tumors are a common form of endocrine neoplasia. However few studies assessed the expression of the principal cyclin regulating checkpoint exit, cyclin D1. Cyclin D1 expression in pituitary tumors and its possible relation to MIB-1 and p27/Kip1 labeling indices (LIs) was explored. DesignWe studied a total of 199 pituitaries, including normal pituitaries (n=7), pituitary adenomas (n=187), and pituitary carcinoma (n=5). All tissues were tested as cores of archived tissue microarrays that were immunostained for cyclin D1, MIB-1 and p27 using a standard technique. Tissue cores were subjected to automated analysis to evaluate the staining LIs. ResultsNo cyclin D1 positive cells in the normal anterior pituitary gland was found. Sparse nuclear staining was noted in pituitary tumors. Higher expression of cyclin D1 was noted in pituitary carcinomas compared to adenomas (p<0.001), in non-functioning adenomas compared to functioning ones (p<0.001) in macroadenomas versus microadenomas (p=0.017) and in recurrent non recurrent adenomas (p<0.001). Cyclin D1 LI and MIB-1 LI were related among adenomas (p<0.001) and carcinomas (p=0.041). p27 LI was neither related to pituitary adenoma recurrence nor invasion. ConclusionsExpression of cyclin D1 in pituitary tumors is related to cell proliferation, recurrence, and metastatic potential. Nuclear cyclin D1 expression is a good marker of aggressive behavior in pituitary tumors.

P21&lt;sup&gt;WAF1&lt;/sup&gt; and hypoxia/reoxygenation-induced premature senescence of H9c2 cardiomyocytes

We have previously reported on hypoxia/reoxygenation-induced premature senescence in neonatal rat cardiomyocytes. In this research, we investigated the effects of p21(WAF1) (p21) in hypoxia/reoxygenation-induced senescence, using H9c2 cells. A plasmid overexpressing wild type p21(WAF1) and a plasmid expressing small hairpin RNA (shRNA) targeting p21(WAF1) were constructed, and transfected into H9c2 cells to control the p21 expression. Hypoxia/reoxygenation conditions were 1% O2 and 5% CO(2), balancing the incubator chamber with N(2) for 6 h (hypoxia 6 h), then 21% oxygen for 8 h (reoxygenation 8 h). Cell cycle was examined using flow cytometry. Senescence was assessed using β-galactosidase staining. The expression of p53, p21, p16(INK4a), and cyclin D1 was assayed using Western blotting. At hypoxia 6 h, cells overexpressing p21 had a larger G1 distribution, stronger β-galactosidase activity, and lower cyclin D1 expression compared to control cells, while the opposite results and higher p53 expression were obtained in p21-knockdown cells. At reoxygenation 8 h, p21-silenced cells had a smaller percentage of G1 cells, weaker β-galactosidase activity and lower 16(INK4a) expression, and higher cyclin D1 expression, but the overexpression group showed no difference. Taken together, this data implies that p21(WAF1) is important for the hypoxia phase, but not the reoxygenation phase, in the H9c2 senescence process.

NKX3.1 contributes to S phase entry and regulates DNA damage response (DDR) in prostate cancer cell lines

NKX3.1 is an androgen-regulated homeobox gene that encodes a tissue-restricted transcription factor, which plays an important role in the differentiation of the prostate epithelium. Thus, the role of NKX3.1 as a functional topoisomerase I activity enhancer in cell cycle regulation and the DNA damage response (DDR) was explored in prostate cancer cell lines. As an early response to DNA damage following CPT-11 treatment, we found that there was an increase in the γH2AX(S139) foci number and that total phosphorylation levels were reduced in PC-3 cells following ectopic NKX3.1 expression as well as in LNCaP cells following androgen administration. Furthermore, upon drug treatment, the increase in ATM(S1981) phosphorylation was reduced in the presence of NKX3.1 expression, whereas DNA-PKcs expression was increased. Additionally, phosphorylation of CHK2(T68) and NBS1(S343) was abrogated by ectopic NKX3.1 expression, compared with the increasing levels in control PC-3 cells in a time-course experiment. Finally, NKX3.1 expression maintained a high cyclin D1 expression level regardless of drug treatment, while total γH2AX(S139) phosphorylation remained depleted in PC-3, as well as in LNCaP, cells. Thus, we suggest that androgen regulated NKX3.1 maintains an active DDR at the intra S progression and contributes to the chemotherapeutic resistance of prostate cancer cells to DNA damaging compounds.

Downregulation of CDKN2A and suppression of cyclin D1 gene expressions in malignant gliomas

BackgroundMalignant gliomas are the most common in central nervous system cancer. Genome-wide association study identifies that CDKN2A was a susceptibility loci for glioma. The CDKN2A/cyclin-dependent kinase 4, 6/Retinoblastoma protein (Rb) pathway is thought to play a crucial role in malignant gliomas pathogenesis. We have investigated the expression of CDKN2A for potential correlations with malignant gliomas grade and potential role of CDKN2A on malignant gliomas pathogenesis.MethodsTumour tissue samples from 61 patients suffering from malignant gliomas were investigated. The expression levels of CDKN2A were detected using immunohistochemical staining and western blot. Overexpression and knockdown of CDKN2A were performed in human glioma cell lines. Subsequently, colony formation, growth curves and CDKN2A-Cyclin-Rb pathway were analyzed.ResultsHere we show that a lower expression of CDKN2A and a higher expression of cyclin D1 in the patients with high-grade malignant gliomas than low-grade gliomas, respectively. Moreover, overexpression of CDKN2A inhibits growth of glioma cell lines by suppression of cyclin D1 gene expression.ConclusionsOur study suggests that CDKN2A as a malignant gliomas suppressor gene, appears to be useful for predicting behaviour of high-grade malignant gliomas. CDKN2A-Cyclin-Rb pathway plays a key role on malignant gliomas formation and that therapeutic targeting of this pathway may be useful in malignant gliomas treatment.

Immunohistochemical and molecular markers in breast phyllodes tumors.

Phyllodes tumors of the breast are diagnostically and managerially enigmatic, as their malignant potential is difficult to predict based on the standard morphologic criteria. Thus, there is a need for additional markers of biologic potential. Although a number of ancillary tests have been reported, consensus in the literature is lacking. We studied 38 cellular fibroadenomas and phyllodes tumors of various grade (World Health Organization benign, borderline, and malignant) with a panel of immunohistochemical stains (p53, CD117, phospho-Histone3, mdm2, cdk4) and screened 26 of the tumors for mutations across 30 cancer-related genes using PCR and mass-spectrometry based methods. p53 and phospho-Histone3 (mitotic marker) showed increased staining in higher grade phyllodes tumors. CD117, mdm2, and cdk4 showed no difference in expression across different grades of phyllodes tumors. Mutational analysis revealed an S8R substitution in FBX4 (an E3 ubiquitin ligase) in 3 cases: 1 benign and 2 borderline. The S8R substitution seems to be more common in phyllodes tumors (11.5%) as compared with other cancers. FBX4 S8R cases had high cyclin D1 expression, but this finding was not specific. Our data support earlier studies showing that p53 has potential use in pathologic assessment of phyllodes tumors, and we newly characterized phospho-Histone3 for this application. Further studies are needed to characterize the molecular pathogenesis of the phyllodes tumors, as we were unable to identify activating mutations despite screening for a large panel of activating hotspot mutations. The significance of the FBX4 substitution deserves further investigation.

41. Differences in cell cycle and apoptotic biomarker expression between molecular subtypes of invasive ductal breast carcinoma

Breast cancer is a heterogeneous disease that can be stratified into at least five subtypes by gene expression analysis or immunohis-tochemical biomarkers. Our aim was to identify differences in the expression of candidate cell cycle and apoptotic biomarkers between these subtypes. We assembled a cohort of 292 patients with early invasive ductal carcinoma and comprehensive clinical follow-up. Using immunohistochemical (ER, PR, CK5/6, EGFR and Ki67) or fluorescent in situ hybridisation (HER2), surrogate signatures for the intrinsic molecular subtypes were defined as follows: Luminal A – ER positive and/or PR positive, HER2 FISH negative, Ki67 low; Luminal B – ER positive and/or PR positive, HER2 FISH positive and/or Ki67 high; HER2 subtype – ER and PR negative, HER2 FISH positive; Basal-like subtype – ER negative, PR negative, HER2 FISH negative, CK 5/6 positive and/or EGFR positive; 5 marker negative – negative for ER, PR, HER2, CK5/6 and EGFR. These signatures were then used to compare the expression of hormone receptors (ER, PR), cell cycle proteins (cyclin D1, cyclin E1, p21WAF1/Cip1, p27Kip) as well as markers of apoptosis (cleaved PARP and TP53) between the molecular subtypes. Luminal A and B subgroups were characterised by a high expression of cyclin D1 (all p The basal-like tumours were characterised by a high cyclin E1 expression (all p These data identify important differences in the expression of a number of cell cycle proteins and TP53 between biomarker-defined breast cancer subtypes. These data may direct further research evaluating the biological aberrations underpinning individual subtypes, their resistance to specific therapies and potential vulnerabilities to new therapeutic approaches.

Abstract P1-04-03: A Role for Cyclin D1 in Neoplastic Transformation in MCF-7 Breast Cancer Cells Post p14ARF-p53 Induced Senescence

Abstract Many years of oncogenic insult and hormonal flux have important long-term implications for breast cancer emergence and progression. Premature-induced senescence is proposed to be a protective response against cancer development. We hypothesize that the senescence response can be subverted in some breast carcinomas, which exhibit latent hormone-dependent proliferative capacity. The ARF-p53 pathway has long been known to play an important role in tumour surveillance halting cell cycle progression to transiently or permanently stop aberrant cells from proliferating. It is well documented that disruption of this pathway is involved in tumourigenesis, in part, through inactivation of cyclin D1. Cyclin D1 plays an important role in breast cancer progression as a regulator of CDK4/6. In addition cyclin D1 has been reported to stimulate estrogen receptor (ER) activity independent of CDK4/6. Our initial observations showed that reactivation of ARF-p53-p21 is indeed a potent rapid inhibitor of cell proliferation in MCF-7 breast cancer cells with a senescent phenotype, however controversially cyclin D1 expression was increased and predominantly located in the nucleus. High expression of nuclear cyclin D1 and p14ARF inversely correlated with cell proliferation. Our studies showed that p14ARF regulated cyclin D1 at the post-transcriptional level. Furthermore, we have evidence from microRNA array analysis that microRNAs known to bind to the cyclin D1 3'UTR sequence were down regulated upon induction of p14ARF, contributing to cyclin D1 stability. We found that long-term exposure to p14ARF was not a failsafe barrier to tumour progression, senescent-like cells re-entering the cell cycle correlated with reduction of nuclear cyclin D1. Conversely, continuous p14ARF expression resulted in chaotic multinucleation and preceded neoplasia. These aberrant nuclei co-stained with cyclin D1 and Ki-67, a well-known marker of cell proliferation. These results suggest that induction of the p14ARF-p53 pathway does not entirely remove the threat of cancer resulting from hyperproliferative oncogenic signals and cyclin D1 plays a promiscuous role in prevention and progression. Arguably, in some breast cancers premature senescent cells may be precursor, or latent cancer cells. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P1-04-03.

Effects of MEK and DNMT inhibitors on arsenic-treated human uroepithelial cells in relation to Cyclin-D1 and p16

Arsenic compounds are well-known toxic and carcinogenic agents, and they are widely distributed throughout the earth's crust. These compounds are associated with various human malignancies. It has been reported that there is an elevated risk of bladder cancer in an area highly contaminated with arsenic on the southwest coast of Taiwan. However, the underlying mechanisms of arsenic-associated carcinogenesis are still unclear. The cell cycle regulatory proteins are important indicators in control of cell cycle progression. Moreover, the high expression of Cyclin-D1 and loss of p16 has been associated with a worse prognosis in a variety of human cancers. Therefore, we investigated the effect of arsenic on Cyclin-D1 and p16 expression and evaluated the role of the ERK signaling pathway and DNA methylation in arsenic carcinogenesis. Our study results showed that Cyclin-D1 high expression was found in 56.3% (9/16) of urothelial carcinomas (UC) from a blackfoot disease (BFD) area and 6.3% (1/16) of UC from a non-BFD area ( p = 0.002). The p16 low expression in 81.2% (13/16) of UC from BFD areas was significantly lower than in non-BFD areas (25.0%; 4/16) ( p = 0.001). In addition, the Cyclin-D1 increased expression but decreased p16 expression in arsenite-treated SV-HUC-1 cells. However, when cells were pretreated with inhibitors (5-aza-CdR or U0126), the effects of arsenite on Cyclin-D1 and p16 expression were suppressed. Finally, these results indicated that Cyclin-D1 and p16 both might play important roles in carcinogenesis as a result of arsenic.

Homeobox transcription factor muscle segment homeobox 2 (Msx2) correlates with good prognosis in breast cancer patients and induces apoptosis in vitro

IntroductionThe homeobox-containing transcription factor muscle segment homeobox 2 (Msx2) plays an important role in mammary gland development. However, the clinical implications of Msx2 expression in breast cancer are unclear. The aims of this study were to investigate the potential clinical value of Msx2 as a breast cancer biomarker and to clarify its functional role in vitro.MethodsMsx2 gene expression was first examined in a well-validated breast cancer transcriptomic dataset of 295 patients. Msx2 protein expression was then evaluated by immunohistochemistry in a tissue microarray (TMA) containing 281 invasive breast tumours. Finally, to assess the functional role of Msx2 in vitro, Msx2 was ectopically expressed in a highly invasive breast tumour cell line (MDA-MB-231) and an immortalised breast cell line (MCF10a), and these cell lines were examined for changes in growth rate, cell death and cell signalling.ResultsExamination of Msx2 mRNA expression in a breast cancer transcriptomic dataset demonstrated that increased levels of Msx2 were associated with good prognosis (P = 0.011). Evaluation of Msx2 protein expression on a TMA revealed that Msx2 was detectable in both tumour cell nuclei and cytoplasm. Cytoplasmic Msx2 expression was associated with low grade tumours (P = 0.012) and Ki67 negativity (P = 0.018). Nuclear Msx2 correlated with low-grade tumours (P = 0.015), estrogen receptor positivity (P = 0.038), low Ki67 (P = 0.005) and high cyclin D1 expression (P = 0.037). Increased cytoplasmic Msx2 expression was associated with a prolonged breast cancer-specific survival (P = 0.049), recurrence-free survival (P = 0.029) and overall survival (P = 0.019). Ectopic expression of Msx2 in breast cell lines resulted in radically decreased cell viability mediated by induction of cell death via apoptosis. Further analysis of Msx2-expressing cells revealed increased levels of p21 and phosphorylated extracellular signal-regulated kinase (ERK) and decreased levels of Survivin and the 'split ends' (SPEN) protein family member RBM15.ConclusionsWe conclude that increased Msx2 expression results in improved outcome for breast cancer patients, possibly by increasing the likelihood of tumour cell death by apoptosis.

Glycodelin expression associates with differential tumour phenotype and outcome in sporadic and familial non-BRCA1/2 breast cancer patients

Glycodelin (encoded by PAEP gene) is a secreted lipocalin protein mainly expressed in reproductive tissues, but also in several tumour types. In the breast, glycodelin is expressed both in normal epithelial and cancerous tissue. To investigate the association of glycodelin with clinicopathological features of breast cancer and outcome of patients we evaluated the protein expression of glycodelin in a large series of breast tumours. Immunohistochemical analysis of tissue microarrays was used to study glycodelin expression on 399 sporadic and 436 familial non-BRCA1/2 tumours with strong family history. Gene expression analysis was used to define genes co-expressed with PAEP in sporadic and familial non-BRCA1/2 breast tumours. In the sporadic series, the glycodelin expression associated with low proliferation rate (P < 0.001), with a tendency towards well-differentiated tumours (grades 1 and 2, P = 0.012) and high cyclin D1 (P = 0.034) expression. However, in familial non-BRCA1/2 cases with strong family history glycodelin expression associated with a less favourable phenotype, i.e. positive lymph node status (P = 0.003) and HER2-positive tumours (P = 0.009). Moreover, the patients with glycodelin-positive tumours had an increased risk for distant metastases (P = 0.001) and in multivariate analysis glycodelin expression was an independent predictor of metastasis (hazard ratio (HR) = 2.22, 95% confidence interval (95% CI) = 1.22-4.03, P = 0.009) in familial non-BRCA1/2 breast cancer. Gene expression analysis further revealed different gene expression profiles correlating with the PAEP expression in the sporadic and familial non-BRCA1/2 breast cancers. Our findings suggest differential progression pathways in the sporadic and familial non-BRCA1/2 breast tumours expressing glycodelin.

Loss of SFRP1 Expression is Associated with Aberrant β-Catenin Distribution and Tumor Progression in Mucoepidermoid Carcinoma of Salivary Glands

Cytoplasmic and nuclear accumulation of beta-catenin in mucoepidermoid carcinoma (MEC) is frequently noted, but the mechanism is unknown. The methylation status of adenomatous polyposis coli (APC) and secreted frizzled-related proteins (SFRPs) was examined by methylation-specific polymerase chain reaction (MSP) assay. The association of SFRP1, beta-catenin, and cyclin D1 expression in MEC was evaluated by immunohistochemical staining. A high percentage of methylation in APC and the SFRP genes was found in MEC compared with adjacent normal tissues, in which SFRP1 (58.6%) was the most frequent methylated gene. Moreover, abundant expression of SFRP1 was noted in normal tissues, whereas reduced SFRP1 expression was detected in 71.7% (33/46) of MECs. There was significant association between methylation and reduced expression of SFRP1. Cytoplasmic/nuclear (C/N) beta-catenin and high cyclin D1 expression were found in 13/55 (23.6%) and 36/55 (65.5%) of cases, respectively. There was significant correlation between C/N beta-catenin expression and reduced SFRP1 expression (P = 0.009). In addition, SFRP1 and beta-catenin expression correlated with tumor malignancy index such as tumor grade and stage. Overall patient survival was significantly worse in patients with reduced SFRP1 and C/N beta-catenin expression (P = 0.009 and P = 0.002, respectively). Methylation of the SFRP1 gene was the major cause of reduced SFRP1 expression. Reduced SFRP1 led to C/N accumulation of beta-catenin and was associated with tumor malignancy. Therefore, examination of SFRP1 expression and beta-catenin location could be useful predictors of tumor progression and prognosis in patients with MEC.

Cyclin D1 is a direct transcriptional target of GATA3 in neuroblastoma tumor cells

Almost all neuroblastoma tumors express excess levels of Cyclin D1 (CCND1) compared to normal tissues and other tumor types. Only a small percentage of these neuroblastoma tumors have high-level amplification of the Cyclin D1 gene. The other neuroblastoma tumors have equally high Cyclin D1 expression without amplification. Silencing of Cyclin D1 expression was previously found to trigger differentiation of neuroblastoma cells. Overexpression of Cyclin D1 is therefore one of the most frequent mechanisms with a postulated function in neuroblastoma pathogenesis. The cause for the Cyclin D1 overexpression is unknown. Here we show that Cyclin D1 overexpression results from transcriptional upregulation. To identify upstream regulators, we searched in mRNA profiles of neuroblastoma tumor series for transcription factors with expression patterns correlating to Cyclin D1. GATA3 most consistently correlated to Cyclin D1 in four independent data sets. We identified a highly conserved GATA3 binding site 27 bp upstream of the Cyclin D1 transcriptional start. Chromatin immune precipitation confirmed binding of GATA3 to the Cyclin D1 promoter. Overexpression of GATA3 induced Cyclin D1 promoter activity, which decreased after site-directed mutagenesis of the GATA3 binding site in the Cyclin D1 promoter. Silencing of GATA3 resulted in reduced Cyclin D1 promoter activity and reduced Cyclin D1 mRNA and protein levels. Moreover, GATA3 silencing caused differentiation that was similar to that caused by Cyclin D1 inhibition. These finding implicate GATA3 in Cyclin D1 overexpression in neuroblastoma.

Correlation of Oncotype DX Recurrence Score with Cyclin D1 and ErbB2.

Abstract Background: The Oncotype DX assay predicts the risk of recurrence in patients with stage I-II ER+, node negative breast cancer treated with tamoxifen. It is not understood if the Oncotype DX assay predicts the natural aggressiveness of an individual breast cancer or if it identifies a subtype of tamoxifen resistant breast cancer. In clinical practice, a high recurrence score (RS) on Oncotype DX is interpreted as a more aggressive tumor and is used to justify the use of chemotherapy. However, if the RS was actually predictive of tamoxifen resistance, patients may benefit from the use of an aromatase inhibitor, and chemotherapy may be unnecessary. Cyclin D1 and ErbB2 are two biomarkers shown to predict tamoxifen resistance.Cyclin D1 is overexpressed in approximately 35% of breast cancers. The Austrian Breast and Colorectal Cancer Study Group assessed expression of Cyclin D1 in patients taking tamoxifen within the ABCSG trial 05 and ABCSG trial 06. In both trials, Cyclin D1 overexpression correlated with a lower relapse free survival and overall survival compared to patients without Cyclin D1 overexpression.Erb2 is overexpressed in 15-30% of breast cancers. In the Gruppo Universitario Napoletano 1 trial, ER+ patients with early stage node negative breast cancer who overexpressed ErbB2 had no improvement in progression free survival and overall survival with 2 years of adjuvant tamoxifen therapy. Additional retrospective studies have supported initial reports of an association between overexpression of ErbB2 and tamoxifen resistance.Methods: 69 patients who had the Oncotype DX assay performed and had unstained pathology slides available were assessed for ErbB2 and Cyclin D1 expression. ErbB2 overexpression status was also obtained in another 74 patients who had the Oncotype DX assay performed. ErbB2 overexpression was determined from a review of medical records where ErbB2 was defined as being positive if immunohistochemical (IHC) staining intensity was 3+ with &amp;gt;90% of cells expressing ErbB2 or FISH revealed an amplification of &amp;gt;2.0. IHC analysis of Cyclin D1 was performed according to standard protocol and using commercially available antibodies. Scoring of slides for Cyclin D1 staining were performed by blinded pathologists who assessed both extent and intensity of nuclear staining for Cyclin D1.Results: The median Oncotype Dx RS within ErbB2+ patients was significantly higher than ErbB2- patients (36.5 vs. 18 p&amp;lt;0.0001), and approximately 50% of patients within each RS grouping (high, intermediate, and low) overexpressed cyclin D1.Conclusion: ErbB2 overexpression among high RS patients suggests the Oncotype DX assay may predict tamoxifen resistance and other markers for tamoxifen resistance need to be correlated with the RS. Although preliminary analysis of the IHC staining for Cyclin D1 does not correlate with a high RS, high Cyclin D1 expression among patients within the low RS subgroup is concerning since this subgroup may have an increased likelihood of disease relapse when treated with adjuvant tamoxifen alone. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3035.

Proteins involved in pRb and p53 pathways are differentially expressed in thin and thick superficial spreading melanomas

Cutaneous melanoma is one of the leading causes of cancer-related death. Malignant transformation of epidermal melanocytes is a multifactorial process involving cell cycle and death control pathways. The purpose of this study was to analyze the immunohistochemical expression of cell-cycle-related and apoptosis-related proteins in cutaneous superficial spreading melanomas using the tissue microarray technique to further understand tumor development. A total of 20 samples of in-situ melanomas and 44 melanomas <or=1.0 mm were analyzed in conventional sections whereas 72 melanomas greater than 1.0 mm and 29 metastases were evaluated by tissue microarray. The sections were stained for the following proteins: p16INK4 (p16), cyclin D1, cyclin-dependent kinase 4 (Cdk4), retinoblastoma protein, tumor suppressor protein p53, and p21 cell cycle regulator (p21) using a streptavidine-biotin-peroxidase technique for immunohistochemistry. Thick melanomas (>1.0 mm) and metastases lost p16 expression in 100% of the cases and in-situ and thin melanomas (<or=1.0 mm) had low rate of p16 expression (7.9%). When comparing thin versus thick melanomas, thin melanomas showed higher expression of cyclin D1 and cytoplasmatic Cdk4, and thick melanomas had increased expression of nuclear Cdk4, tumor suppressor protein p53, and p21. Primary tumors, when compared with metastases, had higher cytoplasmatic Cdk4 expression. None of the studied proteins influenced overall or disease-free survival. Our results suggest that loss of p16 expression was a constant feature in primary and metastatic melanomas. Cyclin D1 expression seems to be related to initial phases of melanoma development. An increase in p21 expression could represent a cell cycle control in proliferating cells with reduced p16 and/or increased nuclear Cdk4 expression.

Cyclin D1 and Cancer Development in Laryngeal Premalignancy Patients

In a previous trial, we found that combined 13-cis-retinoic acid, IFN-alpha, and alpha-tocopherol more effectively reversed advanced premalignant lesions of the larynx than of the oral cavity and that cyclin D1 (CD1) G/A870 single nucleotide polymorphism correlated with cancer risk. We conducted the present trial primarily to confirm the clinical activity of the combination in advanced laryngeal premalignancy and to confirm and extend our findings on CD1, both genotype and protein expression, in association with cancer risk in this setting. Twenty-seven moderate-to-severe laryngeal dysplasia patients underwent induction with combined 13-cis-retinoic acid daily, alpha-IFN twice weekly, and alpha-tocopherol daily for 1 year; 14 nonprogressing patients then were randomized to maintenance fenretinide or placebo for 2 years. During induction, two patients had pathologic complete responses, six had partial responses (30% overall response rate), and five developed laryngeal cancer. There were no significant differences between maintenance fenretinide and placebo in response or cancer rates. Ten patients developed cancer overall. Twenty-four patients were evaluated for the CD1 G/A870 genotype, and 23 for pretreatment and posttreatment CD1 protein expression. Consistent with our earlier report, shorter cancer-free survival was associated with the CD1 AA/AG genotype (P = 0.05). Extending our earlier work, high CD1 expression was associated with worse cancer-free survival overall (P = 0.04) and within each CD1 genotype group. These findings support CD1 genotype and protein expression as important risk markers for laryngeal cancer and suggest future trials targeting upstream regulators of CD1 transcription.