High Levels Of Cyclin D1 Research Articles

BRAF V600E Mutant Colorectal Cancer Subtypes Based on Gene Expression.

Mutation of BRAF at the valine 600 residue occurs in approximately 10% of colorectal cancers, a group with particularly poor prognosis. The response of BRAF mutant colorectal cancer to recent targeted strategies such as anti-BRAF or combinations with MEK and EGFR inhibitors remains limited and highly heterogeneous within BRAF V600E cohorts. There is clearly an unmet need in understanding the biology of BRAF V600E colorectal cancers and potential subgroups within this population. In the biggest yet reported cohort of 218 BRAF V600E with gene expression data, we performed unsupervised clustering using non-negative matrix factorization to identify gene expression-based subgroups and characterized pathway activation. We found strong support for a split into two distinct groups, called BM1 and BM2. These subtypes are independent of MSI status, PI3K mutation, gender, and sidedness. Pathway analyses revealed that BM1 is characterized by KRAS/AKT pathway activation, mTOR/4EBP deregulation, and EMT whereas BM2 displays important deregulation of the cell cycle. Proteomics data validated these observations as BM1 is characterized by high phosphorylation levels of AKT and 4EBP1, and BM2 patients display high CDK1 and low cyclin D1 levels. We provide a global assessment of gene expression motifs that differentiate BRAF V600E subtypes from other colorectal cancers. We suggest that BRAF mutant patients should not be considered as having a unique biology and provide an in depth characterization of heterogeneous motifs that may be exploited for drug targeting. Clin Cancer Res; 23(1); 104-15. ©2016 AACR.

Cdkn2a suppresses metastasis in squamous cell carcinomas induced by the gain-of-function mutant p53(R172H).

p53 (TP53) is the most frequently mutated gene in squamous cell carcinomas (SCCs) of the skin and head and neck. Certain p53 mutations are oncogenic and promote invasion and metastasis in SCCs. However, it is unclear how the oncogenic function of mutant p53 is modulated by other molecular alterations that co-exist in SCCs. Here, we show that deletion of the p53 gene and activation of an endogenous p53(R172H) gain-of-function mutation in the skin induce carcinomas with similar kinetics and penetrance. Deletion of p53 induced primarily well-differentiated SCCs. However, most of the tumours induced by p53(R172H) were poorly differentiated SCCs, the only metastatic tumours in this model. These tumours expressed higher levels of cyclin D1 than the well-differentiated SCCs and spindle carcinomas that developed in these mice. Unexpectedly, metastasis was not observed in mice that developed spindle carcinomas, which expressed high levels of the tumour suppressors p16(Ink4a) and p19(Arf) , encoded by Cdkn2a, a gene frequently deleted in human SCCs. Remarkably, deletion of the Cdkn2a gene in p53(R172H) -induced SCCs promoted a dramatic increase in metastasis rates and a shorter survival in mice that developed these tumours, compared with those observed in mice with tumours in which Cdkn2a was deleted in the presence of a p53 loss-of-function mutation or wild-type p53. Accordingly, the survival of patients with head and neck SCCs bearing co-occurring high-risk p53 mutations and CDKN2A homozygous deletions was much shorter than that of patients with tumours in which high-risk p53 mutations did not contain CDKN2A homozygous deletions, or that of patients with tumours in which homozygous CDKN2A deletions co-existed with either low-risk p53 mutations or potential loss-of-function mutations in p53. These findings genetically identify a population of SCC patients with worst outcomes and will help to predict outcomes according to the p53 status and alterations in CDKN2A. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Increased Wnt Signaling and Reduced Viability in a Neuronal Model of Progranulin-Deficient Frontotemporal Lobar Degeneration.

Progranulin (PGRN) deficiency is considered the major cause of frontotemporal lobar degeneration with TDP-43 protein inclusions (FTLD-TDP). Recent work unveiled a relationship between Wnt signaling and PGRN in cellular models of FTLD and cells of patients carrying loss-of-function GRN mutations. This study was undertaken to explore the relationship between PGRN deficit and Wnt signaling in the regulation of survival of GRN knockdown neuroblastoma SH-SY5Y cells (GRN KD). We report here that both canonical and noncanonical Wnt signaling cascades are overactivated in GRN KD cells. We detected increased expression levels of Wnt1 and Wnt5a ligands of the Frizzled receptors, as well as evidence for increased signaling of the Wnt/β-catenin and Wnt/Ca2+ cascades in PGRN deficient cells, such as increased nuclear content of β-catenin and higher levels of cyclin D1, or increased levels of the active form of the NFAT1 transcription factor, respectively. Upregulation of either Wnt/β-catenin or Wnt/Ca2+ signaling in GRN KD cells leads to the stimulation of BrdU incorporation into DNA, hyperphosphorylation of the pRb family of proteins and reduced cell viability over time. Blocking the Wnt cascades by specific canonical or noncanonical inhibitors of Wnt-dependent signaling, normalized the rate of DNA synthesis, and what it is more important restored the viability of GRN KD cells. Our results suggest an important role of Wnt activation inducing cell cycle disturbance-mediated neuronal loss in the pathogenesis of PGRN deficiency-linked FTLD-TDP. Therefore, it is plausible that modulation of Wnt signaling could be a promising strategy for developing of new disease-modifying treatments for FTLD-TDP.

Cyclin D1 in pediatric neuroblastic tumors: A microarray analysis

Neuroblastoma is the most common extracranial solid childhood tumor, which is believed to originate from primitive neuroblasts giving rise to the sympathetic nervous system. It was previously shown that cyclin D1 (CCDN1) in pediatric neuroblastic tumors (neuroblastoma, ganglioneuroblastoma, and ganglioneuroma) recapitulates its expression during the development of peripheral sympathetic nervous system (PSNS). In the present study, we performed a microarray analysis in order to evaluate the expression of cyclin D1 in neuroblastoma as compared to ganglioneuroma and ganglioneuroblastoma. We first confirmed that comparable levels of cyclin D1 are present in neuroblastoma and fetal neuroblasts. In addition, we observed that neuroblastoma is associated to significantly higher levels of cyclin D1 as compared to both ganglioneuroma and ganglioneuroblastoma. No differences are instead observable between ganglioneuroblastoma and ganglioneuroma. Finally, bioinformatic analysis of cyclin D1-functionally related genes, identified cyclin D2 as an additional marker/etiopathogenic factor in the development of neuroblastoma.

Inhibition of STAT3/cyclinD1 pathway promotes chemotherapeutic sensitivity of colorectal caner

BackgroundChemotherapeutic resistance indicated the poor prognosis of colorectal cancer. ObjectiveOur study aimed to investigate the role of STAT3/cyclinD1 pathway in the chemotherapeutic resistance of colorectal cancer. MethodsWe firstly measured the expression of cyclinD1 in the colorectal cancer tissues using immunohistochemistry in tissue microarray. Then cell viability and apoptosis were investigated in the HT-29 cell lines dealing with recombinant lentivirus and shRNA to increase or decrease cyclinD1 expression. Furthermore, luciferase and ChIP assays were applied to investigate whether STAT3 regulated cyclinD1 expression by binding to its promoter. Finally, we determined whether inhibition of STAT3 could decrease cyclinD1 and increase the chemotherapy sensitivity. ResultsCyclinD1 expression was significantly increased in the cancer cells and high level of cyclinD1 indicated the poor prognosis. Inhibition of cyclinD1 decreased the cell viability assessed by MTT and increased rate of apoptosis when exposed to 5-FU treatment while overexpression of cyclinD1 showed the reverse effect. ChIP assay showed that STAT3 directly bind to cyclinD1 promoter. Subclone of full promoter of cyclinD1 into pGL4 increased the luciferase activity while delete or mutation of any of STAT3 binding sites resulted in reductions of luciferase activity. Inhibition of STAT3 decreased cyclinD1 expression to decrease the cell viability and increase rate of apoptosis when exposed to 5-FU treatment. ConclusionsInhibition of STAT3/cyclinD1 pathway increased the sensitivity of colorectal cancer cell to chemotherapy.

Progesterone Receptor–Cyclin D1 Complexes Induce Cell Cycle–Dependent Transcriptional Programs in Breast Cancer Cells

The progesterone receptor (PR) and its coactivators are direct targets of activated cyclin-dependent kinases (CDKs) in response to peptide growth factors, progesterone, and deregulation of cell cycle inhibitors. Herein, using the T47D breast cancer model, we probed mechanisms of cell cycle-dependent PR action. In the absence of exogenous progestin, the PR is specifically phosphorylated during the G2/M phase. Accordingly, numerous PR target genes are cell cycle regulated, including HSPB8, a heat-shock protein whose high expression is associated with tamoxifen resistance. Progestin-induced HSPB8 expression required cyclin D1 and was insensitive to antiestrogens but blocked by antiprogestins or inhibition of specificity factor 1 (SP1). HSPB8 expression increased with or without ligand when cells were G2/M synchronized or contained high levels of cyclin D1. Knockdown of PRs abrogated ligand-independent HSPB8 expression in synchronized cells. Notably, PRs and cyclin D1 copurified in whole-cell lysates of transiently transfected COS-1 cells and in PR-positive T47D breast cancer cells expressing endogenous cyclin D1. PRs, cyclin D1, and SP1 were recruited to the HSPB8 promoter in progestin-treated T47D breast cancer cells. Mutation of PR Ser345 to Ala (S345A) or inhibition of CDK2 activity using roscovitine disrupted PR/cyclin D1 interactions with DNA and blocked HSPB8 mRNA expression. Interaction of phosphorylated PRs with SP1 and cyclin D1 provides a mechanism for targeting transcriptionally active PRs to selected gene promoters relevant to breast cancer progression. Understanding the functional linkage between PRs and cell cycle regulatory proteins will provide keys to targeting novel PR/cyclin D1 cross talk in both hormone-responsive disease and HSPB8-high refractory disease with high HSPB8 expression.

Estrogen receptor α-coupled Bmi1 regulation pathway in breast cancer and its clinical implications

BackgroundBmi1 has been identified as an important regulator in breast cancer, but its relationship with other signaling molecules such as ERα and HER2 is undetermined.MethodsThe expression of Bmi1 and its correlation with ERα, PR, Ki-67, HER2, p16INK4a, cyclin D1 and pRB was evaluated by immunohistochemistry in a collection of 92 cases of breast cancer and statistically analyzed. Stimulation of Bmi1 expression by ERα or 17β-estradiol (E2) was analyzed in cell lines including MCF-7, MDA-MB-231, ERα-restored MDA-MB-231 and ERα-knockdown MCF-7 cells. Luciferase reporter and chromatin immunoprecipitation assays were also performed.ResultsImmunostaining revealed strong correlation of Bmi1 and ERα expression status in breast cancer. Expression of Bmi1 was stimulated by 17β-estradiol in ERα-positive MCF-7 cells but not in ERα-negative MDA-MB-231 cells, while the expression of Bmi1 did not alter expression of ERα. As expected, stimulation of Bmi1 expression could also be achieved in ERα-restored MDA-MB-231 cells, and at the same time depletion of ERα decreased expression of Bmi1. The proximal promoter region of Bmi1 was transcriptionally activated with co-transfection of ERα in luciferase assays, and the interaction of the Bmi1 promoter with ERα was confirmed by chromatin immunoprecipitation. Moreover, in breast cancer tissues activation of the ERα-coupled Bmi1 pathway generally correlated with high levels of cyclin D1, while loss of its activity resulted in aberrant expression of p16INK4a and a high Ki-67 index, which implied a more aggressive phenotype of breast cancer.ConclusionsExpression of Bmi1 is influenced by ERα, and the activity of the ERα-coupled Bmi1 signature impacts p16INK4a and cyclin D1 status and thus correlates with the tumor molecular subtype and biologic behavior. This demonstrates the important role which is played by ERα-coupled Bmi1 in human breast cancer.

Expression of proliferation markers in colonic mucosa in normal volunteers with a high arginine-containing diet and effect of dietary grape ingestion.

431 Background: Resveratrol, which inhibits ODC and Wnt signaling, has colon cancer prevention activity in animal models. To assess the effects of a resveratrol-rich diet on the colon, 30 normal volunteers enrolled on a cancer prevention clinical trial (NCT00578396) involving the ingestion of 1/3, 2/3, or 1 pound of grapes per day for 14 days. Data analysis was stratified based on arginine intake because meat and dietary arginine have been linked to colon carcinogenesis. Methods: Patients were placed on a low-resveratrol diet 14 days prior to and during the grape ingestion period. Detailed dietary information and colonic biopsies were obtained before and after the grape intervention in a single-subject design. Tissue was processed for RNA for qRT- PCR analysis of genes implicated in colonic proliferation, Wnt signaling and colon carcinogenesis. The % of cells expressing Ki67 in the lower 1/3 of colonic crypts was determined by IHC. Results: Mean arginine consumption for the study population was 4.5 ± 2.27 gm/day. Participants with higher initial dietary arginine consumption (> 4.5gm) had higher levels of cyclinD1 (p = 0.022), c-myc (p = 0.0002), axin2 (p = 0.0065) and CD133 (p = 0.041) mRNA in the colonic mucosa than participants with lower initial arginine consumption. No differences in the % of base of the colonic crypt cells staining for Ki67 were seen. Grape ingestion led to a significant reduction in cyclinD1 expression in participants with higher initial arginine consumption (p = 0.0039), with levels falling to those seen in low arginine consumption participants. Conclusions: Normal volunteers with a high amount of daily arginine ingestion had elevated levels of markers indicative of cell proliferation (cyclinD1, c-myc), Wnt signaling (axin2), and colonic stem cells (CD133). Expression of each of these markers was reduced following grape ingestion, though only the reduction in cyclinD1 reached statistical significance. Significance: Dietary arginine adversely affects colonic mucosa and may predispose individuals to the development of colon cancer. The effects are reversed in part by a resveratrol-rich grape-containing diet. Clinical trial information: NCT00578396.

Cyclin D1 is a major target of miR-206 in cell differentiation and transformation

miR-206, a member of the so-called myomiR family, is largely acknowledged as a specific, positive regulator of skeletal muscle differentiation. A growing body of evidence also suggests a tumor suppressor function for miR-206, as it is frequently downregulated in various types of cancers. In this study, we show that miR-206 directly targets cyclin D1 and contributes to the regulation of CCND1 gene expression in both myogenic and non-muscle, transformed cells. We demonstrate that miR-206, either exogenous or endogenous, reduces cyclin D1 levels and proliferation rate in C2C12 cells without promoting differentiation, and that miR-206 knockdown in terminally differentiated C2C12 cells leads to cyclin D1 accumulation in myotubes, indicating that miR-206 might be involved in the maintenance of the post-mitotic state. Targeting of cyclin D1 might also account, at least in part, for the tumor-suppressor activity suggested for miR-206 in previous studies. Accordingly, the analysis of neoplastic and matched normal lung tissues reveals that miR-206 downregulation in lung tumors correlates, in most cases, with higher cyclin D1 levels. Moreover, gain-of-function experiments with cancer-derived cell lines and with in vitro transformed cells indicate that miR-206-mediated cyclin D1 repression is directly coupled to growth inhibition. Altogether, our data highlight a novel activity for miR-206 in skeletal muscle differentiation and identify cyclin D1 as a major target that further strengthens the tumor suppressor function proposed for miR-206.

Abstract A013: DMP1β, an alternative splice isoform of tumor suppressor hDMP1 locus, has oncogenic properties in breast cancer

Abstract Despite recent advances in therapeutic approaches and early detection, breast cancer remains significant health care burden in many developing countries. In fact, it has been suggested that up to ~30% of breast cancer cases are overdiagnosed as a result of early detection mammography screening. This finding poses a hypothesis that better patient stratification using prognostic/predictive indicators, especially for the patients with early diagnosis, would provide significant improvement in clinical management and reduce health care cost. Our recent work identified Dmp1 as a critical tumor suppressor in breast cancer. Dmp1 is a transcription factor that induces cell cycle arrest and senescence by activating the p14ARF-p53 pathway. Overexpression of Her2/neu activates the Dmp1 promoter via PI3K-Akt-NFκB pathway leading to increase of p53 target genes. Loss of Dmp1 accelerates mammary tumor development in MMTV-neu mouse model without difference between Dmp1+/- and Dmp1-/- genotypes. Human DMP1 locus on 7q21 is hemizygously deleted in ~42% of breast carcinomas, which is mutually exclusive of INK4a/ARF or p53 inactivation. In the cases with hemizygous DMP1 deletion, the other DMP1 allele remains wild-type without mutation or promoter hypermethylation, which suggests that DMP1 is haploinsufficient tumor suppressor. The hDMP1 locus encodes three distinct transcripts via alternative splicing of pre-mRNA at Exon 10. The bona fide tumor suppressor protein is named DMP1α, while the two other transcripts without known biological function were named DMP1β and DMP1γ. The qPCR analysis of 46 matched breast cancer samples revealed that 30% of tumor samples have splicing alteration of DMP1 to increase DMP1β isoform. Importantly, the patients with high DMP1β/α ratio in tumor samples had shorter relapse-free survival compared to those patients without splicing alteration. Furthermore, DMP1β/α ratio was increased in MMTV-neu mouse mammary tumors. Immunohistochemistry of 50 breast tumor samples showed that DMP1β protein is overexpressed which was also associated with shorter relapse-free survival. Expression of DMP1β in non-tumorigenic breast epithelial cell line, MCF10A, significantly increased the rate of cell proliferation and size of the mammospheres in Matrigel©. Knockdown of the endogenous DMP1β inhibits proliferation of breast cancer cell lines. To ascertain role of DMP1β in development of breast cancer in vivo, we developed a MMTV-DMP1β mouse model. The mammary glands from MMTV-DMP1β female mice show dysplastic morphological changes in the epithelium with multifocal tumors at 18 months of age. The tumor tissue and surrounding mammary glands were hyperproliferative as they expressed high levels of Ki67 and Cyclin D1. Overall, we provide evidence that alternative splicing to increase DMP1β expression leads to proliferation of human cells and mouse mammary gland and offers poor prognosis for breast cancer patients. Citation Format: Dejan Maglic, Robert Kendig, Mark Cline, Guangchao Sui. DMP1β, an alternative splice isoform of tumor suppressor hDMP1 locus, has oncogenic properties in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A013.

Heavy Ion Radiation Exposure Triggered Higher Intestinal Tumor Frequency and Greater β-Catenin Activation than γ Radiation in APCMin/+ Mice

Risk of colorectal cancer (CRC) after exposure to low linear energy transfer (low-LET) radiation such as γ-ray is highlighted by the studies in atom bomb survivors. On the contrary, CRC risk prediction after exposure to high-LET cosmic heavy ion radiation exposure is hindered due to scarcity of in vivo data. Therefore, intestinal tumor frequency, size, cluster, and grade were studied in APCMin/+ mice (n = 20 per group; 6 to 8 wks old; female) 100 to 110 days after exposure to 1.6 or 4 Gy of heavy ion 56Fe radiation (energy: 1000 MeV/nucleon) and results were compared to γ radiation doses of 2 or 5 Gy, which are equitoxic to 1.6 and 4 Gy 56Fe respectively. Due to relevance of lower doses to radiotherapy treatment fractions and space exploration, we followed 2 Gy γ and equitoxic 1.6 Gy 56Fe for comparative analysis of intestinal epithelial cell (IEC) proliferation, differentiation, and β-catenin signaling pathway alterations between the two radiation types using immunoblot, and immunohistochemistry. Relative to controls and γ-ray, intestinal tumor frequency and grade was significantly higher after 56Fe radiation. Additionally, tumor incidence per unit of radiation (per cGy) was also higher after 56Fe radiation relative to γ radiation. Staining for phospho-histone H3, indicative of IEC proliferation, was more and alcian blue staining, indicative of IEC differentiation, was less in 56Fe than γ irradiated samples. Activation of β-catenin was more in 56Fe-irradiated tumor-free and tumor-bearing areas of the intestinal tissues. When considered along with higher levels of cyclin D1, we infer that relative to γ radiation exposure to 56Fe radiation induced markedly reduced differentiation, and increased proliferative index in IEC resulting in increased intestinal tumors of larger size and grade due to preferentially greater activation of β-catenin and its downstream effectors.

In vivo RNAi screening identified MKK4 as a new therapeutic target for the improvement of liver regeneration

We established a unique system for performing direct in vivo RNAi screens to genetically dissect cellular signaling networks that regulate proliferation control of hepatocytes during liver damage and regeneration. By combining the well characterized FAH-/- mouse model with a transposon based vector system, we generated chimeric mouse livers. In these mouse livers each hepatocyte expresses FAH, a marker gene and a microRNA based shRNA. To such mouse livers any form of liver damaging protocol can be applied and the regenerative process can be studied. We conducted a first in vivo RNAi screen, using a focused shRNAmir library consisting of 631 constructs targeting 362 genes. Mouse livers were stably repopulated with the shRNAmir library and after repopulation mice were subjected to chronic CCl4 treatment to induce chronic liver damage. The representation of each shRNAmir in the pool, the chimeric mouse liver and after chronic liver damage was determined by deep sequencing analysis. ShRNAs targeting MKK4 showed strong enrichment during liver damage and regeneration. MKK4 belongs to the Map kinase family and is part of two stress kinase signaling pathways, the JNK as well as the p38 pathway. Functional in vivo validation experiments showed that stable knock down of MKK4 by different shRNAs can significantly increase the repopulation efficiency of mouse hepatocytes and also increases the regenerative capacity of chronically damaged mouse livers. In addition we could show that MKK4 suppression also confers an increased robustness of hepatocytes in a model of acute liver failure, resulting in better survival of these animals. Beyond this, primary hepatocytes with stable MKK4 knockdown showed prolonged survival in cell culture and these cells could even be passaged and used to repopulate FAH-/- mouse livers. Protein array and western blot analyses of MKK4 shRNAs versus control-shRNA expressing livers revealed a complex activation pattern of JNK- and p38-signaling downstream factors. Interestingly, the observed pattern does not merely represent an increased JNK over p38 signaling but our data rather suggest that complex cross signaling events between both pathways are involved. Also hepatocytes with stable MKK4 knock down already show higher levels of CyclinD1, suggesting a priming of the cells to leave the G0 state and entering the G1 state of the cell cycle. We developed a new screening platform for pinpointing new regulators of hepatocyte proliferation and liver regeneration. A first in vivo RNAi screen identified MKK4 as an interesting new therapeutic target structure for improving the regenerative capacity under acute and chronic liver damaging conditions.

Attenuation of the Retinoblastoma Pathway in Pancreatic Neuroendocrine Tumors Due to Increased Cdk4/Cdk6

In mice, genetic changes that inactivate the retinoblastoma tumor suppressor pathway often result in pancreatic neuroendocrine tumors (Pan-NETs). Conversely, in humans with this disease, mutations in genes of the retinoblastoma pathway have rarely been detected, even in genome-wide sequencing studies. In this study, we took a closer look at the role of the retinoblastoma pathway in human Pan-NETs. Pan-NET tumors from 92 patients were subjected to immunohistochemical staining for markers of the retinoblastoma pathway. To search for amplifications of retinoblastoma pathway genes, genomic DNAs from 26 tumors were subjected to copy number analysis. Finally, a small-molecule activator of the retinoblastoma pathway was tested for effects on the growth of two Pan-NET cell lines. A majority of tumors expressed high amounts of Cdk4 or its partner protein cyclin D1. High amounts of phosphorylated Rb1 were present in tumors that expressed high levels of Cdk4 or cyclin D1. The copy numbers of Cdk4 or the analogous kinase gene Cdk6 were increased in 19% of the tumors. Growth of the human Pan-NET cell line QGP1 was inhibited in a xenograft mouse model by the Cdk4/6 inhibitor, PD 0332991, which reactivates the retinoblastoma pathway. Inactivation of the retinoblastoma pathway was indicated for most Pan-NETs. Gene amplification and overexpression of Cdk4 and Cdk6 suggests that patients with Pan-NETs may respond strongly to Cdk4/6 inhibitors that are entering clinical trials.

Abstract 4422: Activation of beta-catenin/TCF-4 signaling leading to intestinal tumorigenesis in APCMin/+ mice is radiation quality dependent

Abstract Purpose: Radiation quality depends on loss of energy in tissues per unit distance travelled and could be low-linear energy transfer (low-LET) or high-linear energy transfer (high-LET). The low-LET radiation like gamma-rays and x-rays loose less and hence deposits less energy in tissues and are sparsely ionizing. However, the high-LET radiation like α-particle, neutron, and heavy ions loose more and so deposits more energy in tissues traversed. Epidemiological studies showed strong correlation between radiation exposure and colorectal cancer (CRC) and CRC is the second leading cause of cancer mortality in the USA. However, it is not known if activation of molecular pathways involved in CRC and consequent intestinal tumorigenesis is radiation quality dependent. Methods: Female APCMin/+ mice (6-8 weeks) were irradiated with 5 Gy of gamma radiation, a tumorigenic dose in these mice. After 90 days mice were euthanized, intestinal tumors counted and tumor samples were flash frozen. Tumor samples were subjected to histology, western blot and immunohistochemistry (IHC) to dissect the APC/β-catenin/TCF4 pathway, commonly altered in human CRC. Results of gamma radiation were compared to two qualitatively different types of radiation - high-energy proton (energy: 1 giga electron volt (GeV)) and iron (56-Fe) ion (energy: 1 GeV/nucleon). We used 4 Gy of 56-Fe and 4.7 Gy of proton, which are isotoxic to the gamma radiation dose of 5 Gy determined using a quality factor 1.25 and 1.06 respectively. Quantitation of IHC and western blot were performed using ImageJ v2.45 softwere. Results: Quantitative observations showed enhanced intestinal tumorigenesis after exposure to proton and 56-Fe radiation. Marked increases in β-catenin and TCF4 were associated with a decreased phospho-β-catenin and increased phospho-GSK3β in proton and 56-Fe mice. Increased β-catenin was also associated with higher levels of cyclin-D1 and c-myc in these samples. Furthermore, compared to gamma radiation, strikingly lower level of p53 along with higher MDM2 was observed in proton and 56-Fe samples. Interestingly, however, the decrease in p53 and increase in MDM2 was higher in 56-Fe. These observations were further supported by IHC staining of tumor sections. Statistical analyses to support these observations in proton and 56-Fe compared to ≤ ray or spontaneous tumors will be presented. Conclusions: Considering higher active β-catenin and inactivated GSK3β, we speculate that exposure to high-LET radiation caused stabilization of oncogenic β-catenin and upregulation of c-myc and cyclin-D1 leading to higher tumorigencity. Enhanced potential of high-LET radiation for carcinogenesis is further supported by downregulation of p53. In conclusion, this is the first evidence showing greater potential of high-LET radiation for intestinal carcinogenesis mediated via activation of β-catenin/TCF4 signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4422. doi:1538-7445.AM2012-4422

The impact of cyclin D1 mRNA isoforms, morphology and p53 in mantle cell lymphoma: p53 alterations and blastoid morphology are strong predictors of a high proliferation index

Mantle cell lymphoma is a clinically heterogeneous disease characterized by overexpression of cyclin D1 protein. Blastoid morphology, high proliferation, and secondary genetic aberrations are markers of aggressive behavior. Expression profiling of mantle cell lymphoma revealed that predominance of the 3'UTR-deficient, short cyclin D1 mRNA isoform was associated with high cyclin D1 levels, a high "proliferation signature" and poor prognosis. Sixty-two cases of mantle cell lymphoma were analyzed for cyclin D1 mRNA isoforms and total cyclin D1 levels by real-time reverse transcriptase polymerase chain reaction, and TP53 alterations were assessed by immunohistochemistry and molecular analysis. Results were correlated with proliferation index and clinical outcome. Predominance of the short cyclin D1 mRNA was found in 14 (23%) samples, including four with complete loss of the standard transcript. TP53 alterations were found in 15 (24%) cases. Predominance of 3'UTR-deficient mRNA was significantly associated with high cyclin D1 mRNA levels (P=0.009) and more commonly found in blastoid mantle cell lymphoma (5/11, P=0.060) and cases with a proliferation index of >20% (P=0.026). Both blastoid morphology (11/11, P<0.001) and TP53 alterations (15/15, P<0.001) were significantly correlated with a high proliferation index. A proliferation index of 10% was determined to be a significant threshold for survival in multivariate analysis (P=0.01). TP53 alterations are strongly associated with a high proliferation index and aggressive behavior in mantle cell lymphoma. Predominance of the 3'UTR-deficient transcript correlates with higher cyclin D1 levels and may be a secondary contributing factor to high proliferation, but failed to reach prognostic significance in this study.

Zic2 synergistically enhances Hedgehog signalling through nuclear retention of Gli1 in cervical cancer cells

Aberrant activation of Hedgehog (Hh) signalling has been implicated in the pathogenesis of human cancers. However, the cognate molecular mechanisms contributing to this disregulated pathway are incompletely understood. In this study, we showed that Zic2 was frequently over-expressed and associated with high-grade cervical cancer (p = 0.032), high levels of Gli1 (p < 0.001) and CyclinD1 (p < 0.001) by immunohistochemical and quantitative RT-PCR analyses. Further biochemical studies using luciferase reporter, co-immunoprecipitation, subcellular fractionation and immunofluorescence analyses demonstrated that Zic2 can physically interact with Gli1 and retain it in the nucleus, which in turn increases Gli-mediated transcriptional activity. Gain- and loss-of-function analyses of Zic2 showed that Zic2 could increase Hh signalling activity, cell proliferation and anchorage-independent growth ability in cervical cancer cells. Conversely, deletion of the zinc finger domain at C-terminus of Zic2 significantly abrogated its interaction with Gli1, the retention of Gli1 in the nucleus, effects on Hh signalling activity and oncogenic properties in cervical cancer cells. Our findings suggest that Zic2 is a positive modulator increasing Gli1 transcriptional and oncogenic activity by retaining Gli1 in the nucleus of cervical cancer cells.

A function for cyclin D1 in DNA repair uncovered by protein interactome analyses in human cancers

Cyclin D1 is a component of the core cell cycle machinery. Abnormally high levels of cyclin D1 are detected in many human cancer types. To elucidate the molecular functions of cyclin D1 in human cancers, we performed a proteomic screen for cyclin D1 protein partners in several types of human tumours. Analyses of cyclin D1 interactors revealed a network of DNA repair proteins, including RAD51, a recombinase that drives the homologous recombination process. We found that cyclin D1 directly binds RAD51, and that cyclin D1-RAD51 interaction is induced by radiation. Like RAD51, cyclin D1 is recruited to DNA damage sites in a BRCA2-dependent fashion. Reduction of cyclin D1 levels in human cancer cells impaired recruitment of RAD51 to damaged DNA, impeded the homologous recombination-mediated DNA repair, and increased sensitivity of cells to radiation in vitro and in vivo. This effect was seen in cancer cells lacking the retinoblastoma protein, which do not require D-cyclins for proliferation. These findings reveal an unexpected function of a core cell cycle protein in DNA repair and suggest that targeting cyclin D1 may be beneficial also in retinoblastoma-negative cancers which are currently thought to be unaffected by cyclin D1 inhibition.

Therapeutically targeting cyclin D1 in primary tumors arising from loss of Ini1

Rhabdoid tumors (RTs) are rare, highly aggressive pediatric malignancies with poor prognosis and with no standard or effective treatment strategies. RTs are characterized by biallelic inactivation of the INI1 tumor suppressor gene. INI1 directly represses CCND1 and activates cyclin-dependent kinase (cdk) inhibitors p16(Ink4a) and p21(CIP). RTs are exquisitely dependent on cyclin D1 for genesis and survival. To facilitate translation of unique therapeutic strategies, we have used genetically engineered, Ini1(+/-) mice for therapeutic testing. We found that PET can be used to noninvasively and accurately detect primary tumors in Ini1(+/-) mice. In a PET-guided longitudinal study, we found that treating Ini1(+/-) mice bearing primary tumors with the pan-cdk inhibitor flavopiridol resulted in complete and stable regression of some tumors. Other tumors showed resistance to flavopiridol, and one of the resistant tumors overexpressed cyclin D1, more than flavopiridol-sensitive cells. The concentration of flavopiridol used was not sufficient to down-modulate the high level of cyclin D1 and failed to induce cell death in the resistant cells. Furthermore, FISH and PCR analyses indicated that there is aneuploidy and increased CCND1 copy number in resistant cells. These studies indicate that resistance to flavopiridol may be correlated to elevated cyclin D1 levels. Our studies also indicate that Ini1(+/-) mice are valuable tools for testing unique therapeutic strategies and for understanding mechanisms of drug resistance in tumors that arise owing to loss of Ini1, which is essential for developing effective treatment strategies against these aggressive tumors.

Down-Regulation of the Oncogene Cyclin D1 Increases Migratory Capacity in Breast Cancer and Is Linked to Unfavorable Prognostic Features

The oncogene cyclin D1 is highly expressed in many breast cancers and, despite its proliferation-activating properties, it has been linked to a less malignant phenotype. To clarify this observation, we focused on two key components of malignant behavior, migration and proliferation, and observed that quiescent G(0)/G(1) cells display an increased migratory capacity compared to cycling cells. We also found that the down-regulation of cyclin D1 in actively cycling cells significantly increased migration while also decreasing proliferation. When analyzing a large set of premenopausal breast cancers, we observed an inverse proliferation-independent link between cyclin D1 and tumor size and recurrence, suggesting that this protein might abrogate infiltrative malignant behavior in vivo. Finally, gene expression analysis after cyclin D1 down-regulation by siRNA confirmed changes in processes associated with migration and enrichment of our gene set in a metastatic poor prognosis signature. This novel function of cyclin D1 illustrates the interplay between tumor proliferation and migration and may explain the attenuation of malignant behavior in breast cancers with high cyclin D1 levels.

Loss-of-function mutations in the Transcription Factor 7 (T cell factor-1) gene in hepatogastrointestinal cancers

Inappropriate activation of the Wnt signaling pathway has been repeatedly implicated in the tumorigenesis of colon, liver, and gastric cancers. There is accumulating evidences that transcriptional factor 7 (TCF7; also called T cell factor 1) might also be one of the tumor suppressor genes in the Wnt pathway. We performed PCR-based sequencing analysis of the TCF7 gene in 234 alimentary tract cancers. The TCF7 mutants detected in this study were functionally analyzed after they were generated by a QuickChange site-directed mutagenesis kit. We detected 7 somatic mutations in the TCF7 gene, including 4 missense, 2 frameshift, and one 28-bp deletion. In a yeast two-hybrid assay, most of the mutants showed varying degrees of decreased binding to an amino-terminal enhancer of split (AES), a truncated form of Groucho-related protein lacking WD40 repeats. To determine whether mutant TCF7 proteins had decreased DNA binding, we performed electrophoretic mobility shift assays, and the 2 frameshift mutants were shown to have no DNA binding activity. Furthermore, luciferase reporter assays revealed that TCF7 mutants in the presence of AES failed in the AES-dependent transcriptional repression of the reporter gene. In addition, human embryonic kidney 293 cells transfected with TCF7 mutants expressed high levels of cyclin D1, up to 6 times more than cells transfected with wild-type TCF7. Therefore, the TCF7 mutations detected in this study seem to be “loss-of-function mutations“ caused by loss of TCF7 repressor activity through decreased binding to Groucho-related protein and/or DNA, thereby contributing to neoplastic transformation by causing accumulation of cylin D1.