Amplification Of Cyclin Research Articles

Hepatocellular carcinoma evades RB1-induced senescence by activating the FOXM1-FOXO1 axis.

Hepatocellular carcinoma (HCC) is one of the deadliest cancers. The retinoblastoma protein (RB1), a regulator of cell proliferation, is functionally inactivated in HCC by CYCLIN D/E-mediated phosphorylation. However, the mechanism of RB1-inactivation is unclear because only small percentages of HCCs exhibit amplification of CYCLIN D/E or mutations in the CDK-inhibitory genes. We show that FOXM1, which is overexpressed and critical for HCC, plays essential roles in inactivating RB1 and suppressing RB1-induced senescence of the HCC cells. Mechanistically, FOXM1 binds RB1 and DNMT3B to repress the expression of FOXO1, leading to a decrease in the levels of the CDK-inhibitors, creating an environment for phosphorylation and inactivation of RB1. Consistent with that, inhibition of FOXM1 causes increased expression of FOXO1 with consequent activation of RB1, leading to senescence of the HCC cells, in vitro and in vivo. Also, repression-deficient mutants of FOXM1 induce senescence that is blocked by depletion of RB1 or FOXO1. We provide evidence that human HCCs rely upon this FOXM1-FOXO1 axis for phosphorylation and inactivation of RB1. The observations demonstrate the existence of a new autoregulatory loop of RB1-inactivation in HCC involving a FOXM1-FOXO1 axis that is required for phosphorylation of RB1 and for aggressive progression of HCC.

Phase II trial of palbociclib in patients with advanced esophageal or gastric cancer.

68 Background: Dysregulation of the cell cycle is a hallmark of cancer. Progression through the G1/S transition requires phosphorylation of retinoblastoma (RB) by cyclin-dependent kinases 4 and 6 (CDK4/6), which are regulated by cyclins D and E. A positive feedback loop between apoptosis signal-regulating kinase 1 (ASK1), a member of the MAP kinase pathway, and cyclin D1 has been shown to drive cell proliferation in gastric cancer. In addition, amplification of cyclin D loci and/or activating mutations in CDKs are frequent molecular aberrations in gastroesophageal malignancies. We hypothesized that palbociclib, a potent inhibitor of CDK4/6 would disrupt proliferative signaling, and arrest the growth of gastric cancer. We conducted a phase II trial of palbociclib in gastric and esophageal cancers as an initial test of efficacy. Methods: We screened 38 subjects with gastric, GE junction, or esophageal cancer for RB nuclear expression by immunohistochemistry, and 38/38 (100%) were positive. We enrolled 21 subjects, of whom 5 had gastric adenocarcinoma, 3 had GE junction adenocarcinoma, 8 had esophageal adenocarcinoma, and 5 had esophageal squamous cell carcinoma. Four of 19 subjects tested positive for CCND1 overexpression by FISH. Patients received 125mg daily of palbociclib for days 1-21 of 28-day cycles. Results: Subjects remained on treatment for a median of 1.7 months. By the initial 2-month assessment, 5 of 21 subjects had stable disease, and 16 subjects had progressive disease by imaging and/or clinical progression. No objective responses were seen. The maximum duration of therapy was 5.5 months in two subjects. One of these subjects had progressing HER2-amplified gastric adenocarcinoma, and continued concurrent trastuzumab with palbociclib, while the other had squamous cell carcinoma of the esophagus. Grade 3 or 4 cytopenias occurred in 9 of 21 subjects (43%), with neutropenia in 8 (38%), anemia in 4 (19%), and thrombocytopenia in 1 (5%). One subject discontinued therapy due to grade 4 thrombocytopenia with GI bleed. All other subjects discontinued therapy due to disease progression. Conclusions: Palbociclib has modest single-agent activity in gastroesophageal tumors despite universal RB expression. Clinical trial information: NCT01037790.

Fluorescent peptide biosensor for monitoring CDK4/cyclin D kinase activity in melanoma cell extracts, mouse xenografts and skin biopsies

Melanoma constitutes the most aggressive form of skin cancer, which further metastasizes into a deadly form of cancer. The p16INK4a-Cyclin D-CDK4/6-pRb pathway is dysregulated in 90% of melanomas. CDK4/Cyclin D kinase hyperactivation, associated with mutation of CDK4, amplification of Cyclin D or loss of p16INK4a leads to increased risk of developing melanoma. This kinase therefore constitutes a key biomarker in melanoma and an emerging pharmacological target, however there are no tools enabling direct detection or quantification of its activity.Here we report on the design and application of a fluorescent peptide biosensor to quantify CDK4 activity in melanoma cell extracts, skin biopsies and melanoma xenografts. This biosensor provides sensitive means of comparing CDK4 activity between different melanoma cell lines and further responds to CDK4 downregulation by siRNA or small-molecule inhibitors. By affording means of monitoring CDK4 hyperactivity consequent to cancer-associated molecular alterations in upstream signaling pathways that converge upon this kinase, this biosensor offers an alternative to immunological identification of melanoma-specific biomarkers, thereby constituting an attractive tool for diagnostic purposes, providing complementary functional information to histological analysis, of particular utility for detection of melanoma onset in precancerous lesions. This is indeed the first fluorescent peptide biosensor which has been successfully implemented to monitor kinase activity in skin samples and melanoma tumour xenografts. Moreover by enabling to monitor response to CDK4 inhibitors, this biosensor constitutes an attractive companion assay to identify compounds of therapeutic relevance for melanoma.

Abstract B28: Genomic analysis of osteosarcoma for identification of targeted therapies

Abstract Osteosarcoma (OS) is an aggressive pediatric bone malignancy that affects 400 children per year in the United States. OS is characterized by genomic instability and numerous copy number alterations (CNA). We hypothesized that CNA may be drivers of OS and that targeting cancer genes with CNAs may be a novel approach for treatment of OS. We have established a robust approach for generation of patient-derived xenografts (PDX) from patients with OS. To date, 15 PDX have been established from OS patients. Using whole-genome sequencing (WGS), we have identified possible target CNAs for individual patients and are currently testing them in their corresponding PDX. In two patients, an amplification in c-myc suggested possible susceptibility to the BRD4 inhibitor JQ1 or the CDK9 inhibitor AT7519. In a third patient sample, amplification of Cyclin D and Cyclin E in the setting of wild-type Rb suggested responsiveness to a CDK inhibitor. A fourth patient had a FOXM1 amplification which suggests a vulnerability to Palbociclib, a CDK4/6 specific inhibitor. This patient sample also had an Aurora kinase B amplification, suggesting susceptibility to inhibition of this kinase as a therapeutic approach. We treated the corresponding PDX with the drug predicted to target the patient sample based on CNA. In all cases, treatment of the PDX with the drugs predicted show a significant reduction in tumor growth compared to vehicle alone or to the drugs predicted for the other patient CNAs. As a follow up we are currently submitting several more patient samples and paired PDX for WGS as well as RNAseq to determine other potential targeted therapeutics based on CNA. In summary, our work suggests that WGS can be used to identify potential novel therapies for patients with OS. Further work will be needed to determine whether response in PDX models correlates with clinical response in patients. Citation Format: Leanne Sayles, Marcus Breese, E. Alejandro Sweet-Cordero. Genomic analysis of osteosarcoma for identification of targeted therapies. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B28.

Alterations in the cell cycle checkpoint pathway in breast cancer.

126 Background: Breast cancer is a molecularly diverse disease with molecular subtypes defined by ER, PR and HER2 status. Dysregulation of the cell cycle pathway through activation of cyclin D1 and/or inactivation of CDKN2A is a mechanism of tumor progression in breast cancer and drugs targeting CDK4/6 is undergoing investigation in this disease with promising preliminary results. We investigated several key genetic alterations in cell cycle regulatory proteins in advanced breast cancer patient samples to identify potential targets for cell cycle related treatment options. Methods: A comprehensive genomic profiling was performed on 37 breast cancer samples with the majority being metastatic. Testing included targeted exome sequencing of 562 genes in tumor and paired normal DNA. Results: Patients’ ages ranged from 27-66 years. Analysis of cell cycle regulatory genes revealed focal amplification of cyclin D1/D2 or CDKN2A loss in 9/37 (24%) of cases, of which, 45% were hormone receptor (HR) positive, HER2 negative, 33% were HR positive, HER2 positive and 22% were triple negative (TNBC) subtype. Loss of RB1 was analyzed since it negatively regulates response to CDK4/6 inhibitors. Loss of RB1 was noted in 4/37 (10.8%) of cases, all of which were TNBCs. Only a single TNBC with RB1 loss had concurrent CDK4 amplification and loss of CDKN2A. No evidence of CCNE1 amplification was found in any samples. Incidence of CDK4 and CDK6 amplification was found in samples with cyclin D1 amplification or CDKN2A loss. Our cohort included a single male TNBC patient who harbored cyclin D1 amplification as well as CDKN2A loss. Overall, amplification of cyclin D1, cyclin D2, CDK4 and CDK6 was found in 16%, 5.4%, 2.7% and 2.7% respectively. Loss of CDKN2A was noted in 5.4% of tumors profiled. Conclusions: Exome sequencing identified subsets of patients with mutated cell cycle associated genes that may benefit from CDK 4/6 inhibitors. Our data suggests loss of RB1 is more frequent in TNBC subtype which may confer resistance to CDK4/6 inhibitors. Our analysis suggests that in addition to HR positive, HER2 negative patients, a subset of HR positive, HER2 positive patients could be sensitive to CDK4/6 inhibitors due to activation of the cell cycle pathway as evidenced by mutations in key genes.

Abstract IA18: Tumor suppressor gene silencing by somatic mutations and promoter methylation leads to genomic instability in head and neck squamous cell carcinoma

Abstract IA18: Tumor suppressor gene silencing by somatic mutations and promoter methylation leads to genomic instability in head and neck squamous cell carcinoma

New Strategies for Probing and Inhibiting CDK4/cyclin D Hyperactivity in Melanoma

Uncontrolled proliferation of melanocytes results in melanoma, one of the most aggressive forms of skin cancer, which further metastasizes into a deadly form of cancer. The p16‐cyclin D‐CDK4/6‐retinoblastoma protein pathway is dysregulated in 90% of melanomas and activation of this pathway potently cooperates with mutant BRAF or NRAS in transformation of melanocytes. CDK4/cyclin D kinase hyperactivation, associated with mutation of CDK4, amplification of cyclin D or loss of p16INK4a leads to increased risk of developing melanoma. This kinase therefore constitutes a key biomarker and an emerging pharmacological target.The limited efficacy of current therapeutics for treatment of melanoma and the lack of diagnostic assays to detect the early onset of this disease call for new generations of targeted therapeutics and novel approaches to probe melanoma‐specific biomarkers. To this aim, we engineered a fluorescent biosensor which reports on CDK4 kinase hyperactivity in vitro and in living cells, which we employed to develop a diagnostic assay using human skin biopsies. We further designed biosensors for drug discovery purposes, which discriminate compounds that target the interface between CDK4 and cyclin D, or that interfere with conformational dynamics of CDK4 activation loop, which were successfully applied to screen for novel and potent classes of CDK4 inhibitorsOur work highlights the potency of fluorescent biosensors for developing diagnostic strategies to monitor cancer‐associated alterations in CDK4 activity as well as drug discovery programmes.

Cyclin alterations in diverse cancers: Outcome and co-amplification network.

Cyclin genes are key regulatory components of the cell cycle. With the development of new agents, cyclin-related genes are becoming increasingly important as they can be targeted. Yet, the biological implications of these alterations have not been fully studied. Clinical characteristics and outcome parameters were compared for patients harboring cyclin alterations versus not. CCN alterations were found in 13% of our population (50/392; all amplifications) and were associated with breast cancer (P < 0.0001), a higher median number of concomitant molecular alterations (P < 0.0001), and liver metastases (P = 0.046). Harboring a cyclin amplification was not associated with overall survival, the time to metastasis/recurrence, nor with the best progression-free survival. In a Cox regression model, gastrointestinal histology (P < 0.0001), PTEN (P < 0.0001), and CDK alterations (P = 0.041) had a significant association with poorer overall survival. CCN amplifications significantly correlated with alterations in FGF/FGFR family genes as well as in MET and ARFRP1. An extended correlation study shed light on a network of co-amplifications influenced in part by genes that were localized on the same amplicons. CCN amplifications are common across cancers and had distinctive biological associations. Customized combinations targeting the cyclin pathway as well as the extended co- amplification network may be necessary in order to address resistance mechanisms.

Abstract SY34-03: Braking the cycle: Inhibition of the cyclin D-Cdk4/6 pathway in breast cancer

In spite of the dramatically improved outlook for patients with ER+ breast cancer, a subset of patients are intrinsically non-responsive to anti-hormonal therapy alone, and even the responders invariably acquire resistance to this treatment modality. In the setting of established hormone resistance, most of these patients will receive chemotherapy, with limited benefit and considerable impact on quality of life due to toxicity. Given the sheer numbers of patients affected (approximately two-thirds of all breast cancers are ER+), the unmet need for well-tolerated therapies that overcome resistance to anti-hormonal agents remains very high. Strikingly, the vast majority (>90%) of ER+ breast cancers express a functional retinoblastoma protein (Rb), a tumor suppressor that represents a critical regulator of the G1 to S phase transition in mammalian cells. In its hypo-phosphorylated state, Rb suppresses the expression of proteins that are essential for commitment to S-phase entry and progression through the remainder of the eukaryotic cell cycle. The G1 cyclin-dependent kinases 4 and 6 (cdk4 and cdk6) which function in complexes with the D-type cyclins (collectively termed cyclin D) initiate the phosphorylation of Rb and override the repressive effects of Rb on cell-cycle progression. In ER-driven breast cancers, oncogenic signaling through the ER stimulates the cyclin D-cdk4/6 -dependent phosphorylation of Rb, and this proliferative stimulus is frequently augmented by amplification of cyclin D or loss of expression of the cyclin D-cdk4/6 inhibitor, p16 (encoded by the CDKN2A gene), two alterations that lead to elevated and dysregulated cyclin D-cdk4/6 activity. The requirement for cyclin D-cdk4/6 activity to circumvent the cell-cycle braking action of Rb suggested that ER+ breast cancer cells might be vulnerable to drug-induced inhibition of cdk4/6 activity. PD-0332991 is an orally administered, selective inhibitor of cyclin D-cdk4/6 kinase activities. In preclinical models, this drug showed significant anti-proliferative activity in Rb+ breast cancer cell lines, the majority of which also expressed the ER. The most well established substrate for the cyclin D-cdk4/6 is Rb, which is modified at multiple phosphoacceptor sites (including Ser-780, Ser-795, Ser-807, and Ser-811) by these cdks. The phosphorylation status of the Ser-807 and Ser-811 residues in Rb proved to be the most robust pharmacodynamic readouts for PD-0332991 activity in vivo, and serve as appropriate biomarkers for target modulation by this drug under in vitro and in vivo study conditions. The IC50 for reduction of Rb phosphorylation at these sites in two breast cancer cell lines (MCF7 and T47D) was approximately 20 nM. Similarly, we observed that PD-0332991 inhibits S-phase entry (measured by incorporation of thymidine into newly synthesized DNA) and cellular proliferation in panel of Rb+ cancer cell lines, with IC50 values ranging from 40-170 nM. The antiproliferative effects of PD-0332991 were not limited to transformed cells, reinforcing the idea that this drug was capable of suppressing the proliferation of any human cell type that requires cyclin D-cdk4/cdk6 activity to bypass the G1 restriction point imposed by Rb. Conversely, cancer cells that have inactivated Rb through genetic or epigenetic mechanisms are highly resistant (IC50 > 3 uM) to the inhibitory effects of PD-0332991 on cell-cycle progression and cellular proliferation. Xenograft experiments confirmed the favorable in vivo safety profile and antitumor activity of PD-0332991. The maximum tolerated dose (MTD; defined as the dose that induced These preclinical studies provided a launching pad for a clinical trial in post-menopausal women with locally advanced or metastatic ER+ (HER2-) breast cancer. In this Phase 2 study, PD-0332991 was given in combination with letrozole (an inhibitor of estrogen synthesis), with the control arm receiving letrozole only. An interim analysis showed that the PD-0332991 plus letrozole combination yielded a dramatic improvement in treatment outcomes, more than tripling progression-free survival relative to the letrozole-only treatment arm. The drug was generally well-tolerated, due in part to its high degree of selectivity for cdk4/6 relative to other members of the cyclin-dependent kinase family, as well as other members of the extended kinase superfamily. These positive results led to a Breakthrough Therapy designation in 2013 for PD-0332991 in the treatment of patients with ER+ breast cancer. This presentation will provide information on the mechanisms of antitumor action of PD-0332991, together with updates on the current status of clinical development of this new addition to the armentarium of breast cancer treatments. Citation Format: Robert T. Abraham, Todd VanArsdale, David V. Shields, Nathan V. Lee, Maria Koehler, Kim Arndt. Braking the cycle: Inhibition of the cyclin D-Cdk4/6 pathway in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr SY34-03. doi:10.1158/1538-7445.AM2014-SY34-03

Molecular screening for cancer treatment optimization (MOSCATO 01): A prospective molecular triage trial—Interim results.

2512 Background: Characterization of the genomic alterations (GA) that could drive tumor growth of an individual patient (pt) is now critical to better select targeted therapies in phase I trials. Methods: Pts with advanced solid tumors, who failed at least one line of standard therapy, were offered an on-purpose tumor biopsy for molecular characterization. Biopsies were mainly obtained using 18G needles under CT or ultra-sound control, from metastatic or primary tumor sites. DNA extracted from fresh tumor biopsies was analyzed by CGH (Agilent platform) (if ≥ 50% tumor cells in the sample) and by sequencing for 30 target genes (if ≥ 30% tumor cells in the sample). An expert panel of scientists and clinicians reviewed results to determine the biological signification of the GA and match such pts to the most relevant targeted therapy available (mainly in early clinical trials). PFS using therapy based on GA was compared to the PFS for the most recent therapy on which the pt had just experienced progression (PFS ratio). Results: From December 2011 to august 2012, 129 heavily pretreated pts (median of 3 previous lines) were consented. Among them, 111 (86%) had a dedicated tumor biopsy. An actionable target was identified in 52 patients (40%). Among them, 25 pts (23% of biopsied pts) have been treated with a targeted therapy. The median time between biopsy and molecular results was 21 days [17 – 28]. GA of interest encompassed FGF ligand or receptor amplification (n=9), cyclin amplification or deletion (n=4), KRAS/BRAF/NRAS mutation (n=3), PI3K amplification or PTEN deletion (n=3), EGFR amplification or mutation (n=3) (outside lung cancer), HER2 amplification (n=2) (outside breast cancer), EML4/ALK translocation (n=1). Among the 25 pts treated according to their GA, we observed 5 PR (20%), 14 SD (56%) and 3 PD (12%). Three pts (12%) were not evaluable because of early discontinuation of the therapy. PFS ratio was &gt;1.3 among 9 out of 19 evaluable pt (47%). Conclusions: High throughput molecular analysis is feasible in daily practice. It allows enrichment of phase I trials with specific GA, and leads to promising anti-tumor activity (20% PR as compared to the classical 7-10% PR obtained in all comers phase I trials). Clinical trial information: NCT01566019.

Does more intensive therapy have effects on mantle cell lymphoma? A clinical experience from the Lymphoma Treatment Study Group in Japan

Mantle cell lymphoma (MCL) is a rare subtype of B-celllymphoma characterized by amplification of cyclin D1.The effect of chemotherapies, such as CHOP (cyclophos-phamide, doxorubicin, vincristine, and prednisone), is oftentransient, and most patients die at median 3–5 years fromdiagnosis [1]. Before the rituximab era, a number of smallstudies evaluated the efficacy of high-dose chemotherapy(HDC) supported with autologous stem cell transplantation(ASCT), but did not find an evident contribution to survivalbenefit [2, 3]. Consequently, no single standard treatmentfor MCL has been established to date [4]. We thereforeconducted a nation-wide multicenter retrospective analysisof newly diagnosed patients with MCL in order to inves-tigate the efficacy and clinical outcome of each initialtreatments and HDC/ASCT in the rituximab era.Clinical data of 64 newly diagnosed MCL cases treated in12 institutions participating in the Lymphoma TreatmentStudyGroupinJapanbetween2001and2008werecollectedand analyzed by the end of April 2009. The data includedpatient characteristics [age, sex, performance status definedby European Cooperative Oncology Group (ECOG PS),clinical stage according to the Ann Arbor staging system,complete blood count, and serum lactate dehydrogenaselevel], as well as details of treatment and clinical outcome.Positivity of cyclin D1 and/or chromosomal abnormalityt(11;14)(q13;q32) in the biopsy specimen were required forthe diagnosis of MCL following the World Health Organi-zation classification system. This study was approved by theinstitutional review board in Nihon University ItabashiHospital, where the analysis was performed.Table 1 shows the clinical characteristics of all patientsand risk factors that might influence overall survival,including MCL international prognostic index (MIPI) [5].The median age of patients and median observation periodwere 64 years (range 47–80 years) and 34 months (range2–91 months), respectively. Most of patients were treatedwith rituximab-combined CHOP (R-CHOP)-based che-motherapy for a median 6 (range 2–8) cycles or R-Hyper-CVAD/MA regimen (rituximab, hyperfractionatedcyclophosphamide, vincristine, doxorubicin, and dexameth-asone alternating with high-dose methotrexate and cytara-bine), as initial treatment. As a result, 5-year overallsurvival (OS) and progression-free survival (PFS) for allpatients were 62 and 34%, respectively (Fig. 1a). Thecomplete response rate of the R-CHOP group was lowerthan that of the R-Hyper-CVAD/MA group (49 vs. 80%,P = 0.031), but there was no significant difference in OSor PFS (Fig. 1b, c). Sixteen patients (25%) received vari-ous combinations of HDC followed by ASCT. Amongthese patients, nine underwent HDC/ASCT in their firstcomplete remission, six were in a refractory phase of thedisease, and one was in a second remission. The clinicaloutcome of this group was quite encouraging, withouttreatment-related mortality or secondary malignancy dur-ing the observation period (93% of 5 year OS) (Fig. 1d).In this study, the majority of patients were treated withR-CHOP-based chemotherapy. After rituximab becameclinically available for treating B-cell lymphomas, theclinical outcome of MCL appeared to be improved.

Genome wide SNP comparative analysis between EGFR and KRAS mutated NSCLC and characterization of two models of oncogenic cooperation in non-small cell lung carcinoma

BackgroundLung cancer with EGFR mutation was shown to be a specific clinical entity. In order to better understand the biology behind this disease we used a genome wide characterization of loss of heterozygosity and amplification by Single Nucleotide Polymorphism (SNP) Array analysis to point out chromosome segments linked to EGFR mutations. To do so, we compared genetic profiles between EGFR mutated adenocarcinomas (ADC) and KRAS mutated ADC from 24 women with localized lung cancer.ResultsPatterns of alterations were different between EGFR and KRAS mutated tumors and specific chromosomes alterations were linked to the EGFR mutated group. Indeed chromosome regions 14q21.3 (p = 0.027), 7p21.3-p21.2 (p = 0.032), 7p21.3 (p = 0.042) and 7p21.2-7p15.3 (p = 0.043) were found significantly amplified in EGFR mutated tumors. Within those regions 3 genes are of special interest ITGB8, HDAC9 and TWIST1. Moreover, homozygous deletions at CDKN2A and LOH at RB1 were identified in EGFR mutated tumors. We therefore tested the existence of a link between EGFR mutation, CDKN2A homozygous deletion and cyclin amplification in a larger series of tumors. Indeed, in a series of non-small-cell lung carcinoma (n = 98) we showed that homozygous deletions at CDKN2A were linked to EGFR mutations and absence of smoking whereas cyclin amplifications (CCNE1 and CCND1) were associated to TP53 mutations and smoking habit.ConclusionAll together, our results show that genome wide patterns of alteration differ between EGFR and KRAS mutated lung ADC, describe two models of oncogenic cooperation involving either EGFR mutation and CDKN2A deletion or cyclin amplification and TP53 inactivating mutations and identified new chromosome regions at 7p and 14q associated to EGFR mutations in lung cancer.

The Prognostic Value and Overexpression of Cyclin A Is Correlated with Gene Amplification of both Cyclin A and Cyclin E in Breast Cancer Patient

Deregulation of cell cycle control is a hallmark of cancer. The primary cyclins (A, B1, D1, D3 and E) are crucial for cell cycle progression. Secondary cyclins (C and H) have putative indirect effects on cell cycle propulsion and are not previously evaluated in breast cancer. We have examined protein expression and gene amplification of cyclins in breast carcinomas and correlated the findings with clinical follow-up data. We have previously demonstrated that over-expression of cyclin A is associated with poor prognosis in breast cancer patients. In this study we wanted to evaluate the mechanisms behinde overexpression of cyclin A, as well as the impact of other cyclins, both at the gene level and at the protein level, on prognosis of breast cancer patients. The impact of TP53 gene mutations on gene amplification of cyclins was also evaluated. Methods: Real-Time Quantitative PCR was used to detect gene amplification of cyclins in tumour tissue from 86 patients operated for invasive breast carcinomas, while immunohistochemistry was applied to detect protein expression of the same cyclins. Result: Of the 80-breast tumour samples available for cyclin A gene amplification analyses, 26.7% (23/80) was defined to have cyclin A gene amplification. 37.2% (32/79) had cyclin B1 gene amplification, 82.6% (71/82) of the samples harboured amplification of cyclin C gene, 74.4% (64/82) had cyclin D1 gene amplification, 41.9% (36/86) had cyclin D3 gene amplification, 29.1% (25/81) of the patients had cyclin E gene amplification and 9.3% (8/86) of the samples showed amplification of the cyclin H gene. When correlation between gene amplification and protein expression was evaluated, we observed a statistical significant correlation between gene amplification and protein expression of cyclin A (p = 0.009) and cyclin D3 (p < 0.001). However, the correlation between gene amplification and protein expression of cyclin A, as well as the prognostic value of cyclin A overexpression, was affected by gene amplification of cyclin E. Gene amplification of none of the other cyclins was associated with patient prognosis. There was a statistical significant correlation between TP53 gene mutations and gene amplification of cyclins A, D3 and B1. No correlation was observed between gene amplification of secondary cyclins (H and C) and TP53 gene mutations. Conclusions: The overexpression of cyclin A is correlated to gene amplification of both cyclin A and cyclin E. Over-expression of cyclin A is associated with poor prognosis in breast cancer patients. When analysed in a multivariate analyses model, gene amplification as well as protein expression of none of the other cyclins than cyclin A are associated with patient prognosis in breast carcinomas. TP53 gene mutation seems to correlate with gene amplification of primary, but not secondary cyclins.

Protein expression and gene amplification of primary cyclins (A, B1, D1, D3and E) and secondary cyclins (C and H) in relation to prognosis in breast cancer patients

Deregulation of cell cycle control is a hallmark of cancer. The primary cyclins (A, B1, D1, D3 and E) are crucial for cell cycle progression [1]. Secondary cyclins (C and H) have putative indirect effects on cell cycle propulsion and have not been previously evaluated in breast cancer. We have examined protein expression and gene amplification of cyclins in breast carcinomas and correlated the findings with clinical follow-up data. We have previously demonstrated that overexpression of cyclin A is associated with poor prognosis in breast cancer patients [2]. In this study we wanted to evaluate the impact of other cyclins, both at the gene level and at the protein level. We wanted to evaluate whether the overexpression of cyclins is a result of gene amplification, as well as to evaluate the prognostic value of gene amplification of different cyclins for breast cancer patients. The impact of TP53 gene mutations on gene amplification of cyclins was also evaluated. Real-time quantitative PCR was used to detect gene amplification of cyclin A, cyclin B1, cyclin C, cyclin D1, cyclin D3, cyclin E and cyclin H in tumour tissue from 80 patients operated for invasive breast carcinomas, while immunohistochemistry was applied to detect protein expression of the same cyclins. Among the 80 breast cancer tumour samples examined, 26.7% was defined to have ccnA2 gene amplification, 37.2% had ccnB1 gene amplification, 82.6% of the samples harboured amplification of ccnC, 74.4% had ccnD1 gene amplification, 41.9% had ccnD3 gene amplification, 29.1% of the patients had ccnE gene amplification and 9.3% of the samples showed amplification of the ccnH gene. When correlation between gene amplification and protein expression was evaluated, we observed a statistical significant correlation between gene amplification and protein expression of cyclin A (correlation coefficient = 0.287, P = 0.009) and cyclin D3 (correlation coefficient = 0.906, P = 4.9 × 10-33). Protein expression as well as gene amplification of cyclin A was also correlated with gene amplification of other cyclins. When the impact of gene amplification of different cyclins on the patient survival was analysed, only gene amplification of cyclin A was associated with patient survival. We found a significant interaction between amplification of cyclin A and cyclin E (Cox regression, P = 0.02). These two cyclins are sequentially time related in the cell cycle. The effect of amplification of cyclin A was therefore tested in a stratified analysis both when the cyclin E gene was not amplified and when the cyclin E gene was amplified. When the cyclin E gene was not amplified, the statistical strength of the cyclin A amplification increased with a HR of 5.5 (95% confidence interval: 2.2–14.3, P < 0.0001). When cyclin E was amplified, amplification of cyclin A had no significant impact on survival (P = 0.45). In summary, we have analysed gene amplification and protein expression of both primary and secondary cyclins in invasive breast carcinomas. Overexpression and gene amplification of cyclin A is correlated with gene amplification of other cyclins. Only gene amplification and overexpression of cyclin A was associated with poor prognosis, and amplification of cyclin A is the strongest prognostic factor in patients that have a normal amplicon of cyclin E.

Expression and gene amplification of primary (A, B1, D1, D3, and E) and secondary (C and H) cyclins in colon adenocarcinomas and correlation with patient outcome

Background/Aims: Deregulation of cell cycle control is a hallmark of cancer. The primary cyclins (A, B1, D1, D3, and E) are crucial for cell cycle progression. Secondary cyclins (C and...

Reciprocal expressions of cyclin E and cyclin D1 in hepatocellular carcinoma

Deregulation of the cell cycle by overexpression of G1 cyclins, cyclin E and cyclin D1 genes, has been demonstrated to be a prerequisite for the development of human cancer. Recently, cyclin E is proposed to be sufficient for the progression of the G1 cell cycle without cyclin D1. Here we show that the proposed model system was specifically present in human hepatocellular carcinoma (HCC) unlike other human cancers. Of 31 HCC tissues analyzed, 21 (67.7%) exhibited an overexpression of cyclin E protein. In contrast to cyclin E gene expression, cyclin D1 expression was strongly downregulated in 19 (61.2%) HCCs. Interestingly, 65% of HCC tissues with overexpression of the cyclin E gene exhibited downregulation of cyclin D1, suggesting reciprocal deregulation of these cyclins in the G1 progression of the cell cycle. Southern blot analysis proved the amplification of cyclin E gene in HCC with a high level of overexpression. The present findings suggest that the reciprocal deregulation of cyclin E lacking cyclin D1 expression might play a role in G1 progression and the development of HCC.

Genetic marker analysis in head and neck cancer

Prognostication of head and neck cancer (HNCC) involves molecular identification of residual tumor cells, prediction of recurrence, distant metastases or secondary tumors and prediction of the sensitvity to therapy. Biomarkers of HNCC are mutations of p53, p16 and amplification of Cyclin D and E2F4. One hundred and fifty-two HNCC cases have been evaluated for p53, hMLH1, Cyclin D and p16 gene alterations using PCR-SSCP and Western blot analysis. P53 mutations of HNCC have been found in 37.5% of cases. However, 11% of the cases showed p53 mutations in the normal peritumoral mucosa suggesting "field cancerization" process. Mismatch-repair gene mutations (MMR: hMHL1 and hMSH2) occurred with 17 and 8.6% frequency, respectively, while E2F4 mutations were even more frequent (21.4%) in HNCC. Our data suggest that E2F4 overexpression can be caused by the inactivation of the p16 gene in HNCC, while its mutations are most probably associated to the mutations of the MMR genes. These molecular informations can help to predict the biological potential of HNCC as well as the probability of the development of secondary HNCCs.

Cell cycle control as a basis for cancer drug development (Review).

Normal cell cycle progression relies on the cell's ability to translate extracellular signals such as mitogenic stimuli and intact extracellular matrices in order to efficiently replicate DNA and divide. Cyclin dependent kinases (cdks) respond to these signals and are largely responsible for positively pushing cells through the cell cycle. Due to their pivotal role in cell division, nature has evolved elaborate mechanisms to regulate the kinase activity of cdks. Cyclins are cdk binding partners which are required for kinase activity and their protein levels are intimately linked to the cell cycle stage. A variety of other cdk regulators such as phosphorylation events, natural inhibitors and complex stability are discussed. Phosphorylation of various cdk substrates results in diverse outcomes such as changes in gene expression, formation of prereplicative complexes and breakdown of the nuclear envelope. Cancer cells evolve in part by over-riding normal cell cycle regulation. Abnormal cdk activity is accomplished by cyclin amplification, cdk or substrate mutation as well as inactivation of inhibitors. The selective growth advantage of cancer cells also stems from amplification of positive growth signals, mutation of checkpoint and surveillance genes as well as deregulation of programmed cell death or apoptosis. The full potential of cancer therapies, such as small molecule inhibitors and gene therapy among others, focusing on our knowledge of cell cycle regulation has yet to be reached.

Amplification of the cyclin A gene in canine and feline mammary tumors.

DNAs from 33 canine mammary tumors and 8 feline mammary carcinomas were examined by Southern blot analysis to clarify genomic abnormalities of the cyclin A gene. Amplification of cyclin A was detected in 27.3% (9/33) of canine mammary tumors and 87.5% (7/8) of feline mammary carcinomas. It was suggested that amplification of cyclin A do not correlate directly with the tumorigenesis of canine mammary tumors, because there was no significant difference of incidence of cyclin A amplification between the benign and malignant tumors. In feline mammary carcinomas, the high frequency of cyclin A amplification raised the possibility that the amplification lead to the protein overexpression and play an important role in the tumorigenesis.

Inhibition of cell proliferation in head and neck squamous cell carcinoma cell lines with antisense cyclin D1

Cyclin D1 and cyclin G are essential regulatory factors in the progression of the cell cycle from G0 through G1 and S phase. Aberrations in expression of these cyclins may lead to dysregulated cellular proliferation that could result in neoplasia. Amplification and overexpression of cyclin D1 have been observed in many human cancers, whereas cyclin G is a new cyclin recently described in osteosarcoma cells. This study was performed to determine whether these cyclins were amplified in head and neck squamous cell carcinoma (HNSCC) tumors. Polymerase chain reaction of DNA extracted from 22 HNSCC primary tumors and three HNSCC cell lines did not reveal amplification of cyclin D1 in any of the tumor samples. Southern blot analysis identified amplification of cyclin D1 in a single tumor. Amplification of cyclin G was not observed in any of the tumors by Southern blot hybridization with a cyclin G probe. HNSCC cell lines transfected with antisense cyclin D1 were tested for cell proliferation by the incorporation of 3 H-thymidine into cells grown in serum-free media. By 72 hours of incubation, there was a greater than 30% reduction in proliferation of cells transfected with antisense cyclin D1 as compared with nontransfected control cells. The results indicate that cyclin D1 may play an important role in the growth and proliferation of HNSCC cells. (Otolaryngol Head Neck Surg 1998;119:593-9.)