Cyclin D1 Locus Research Articles

SAT177 Molecular Analysis Of AMBRA1 As A Candidate Tumor Suppressor In Sporadic Parathyroid Adenomas

Abstract Disclosure: S. Chinwo: None. J. Costa: None. A. Arnold: None. J. Bellizzi: None. Primary hyperparathyroidism is a common endocrine disorder that is most often caused by a sporadic single-gland parathyroid adenoma. Currently, the only known and experimentally validated oncoprotein for parathyroid adenomas is cyclin D1. The overexpression of cyclin D1 protein has been reported in 20-40% of cases and about 8 percent of cases harbor a DNA rearrangement involving the cyclin D1 (CCND1) locus. Otherwise, the molecular mechanisms underlying this cyclin D1 overexpression have yet to be identified. Here, we explored a potential parathyroid tumorigenic mechanism that could increase cyclin D1 stability through a defect in the AMBRA1 E3 ligase adaptor, a molecule responsible for its degradation. Evidence that AMBRA1 may be a tumor suppressor gene includes the accumulation of cyclin D with hyperproliferation in AMBRA1-deficient cells, the context-dependent growth of tumors in Ambra1-deficient mice, and the finding that low levels of AMBRA1 correlated with poor clinical outcomes in The Cancer Genome Atlas. Therefore, we proceeded to examine AMBRA1 for evidence of tumor suppressor-type inactivation within a cohort of 98 typical parathyroid adenomas. Genomic DNA samples from these tumors were subjected to PCR-amplification of all 18 coding exons of AMBRA1, followed by Sanger sequencing. Sequences were analyzed by comparison to the normal reference sequence: ENST00000683756.1, seeking to assess any observed intragenic or splice-site variants as potential or likely loss-of-function mutations. Thus far, with 97 percent of the coding region for this 1298-amino acid protein fully sequenced, we have identified an inactivating mutation in 1 of the 98 tumors (1%): c.126G>A in exon 2, resulting in an early stop codon p.Trp42*. Sequencing the patient’s matched germline DNA confirmed the mutation was somatic and showed loss of heterozygosity. We also identified likely-nonpathogenic variants in 5 parathyroid adenomas: missense variant c.3385G>T (p.Ala1129Ser), 3’ UTR variants c.*576A>T and c.*491T>C and synonymous variants c.579C>T and c.2776C>T, each in a single tumor. 4 of 5 were found as germline variants in the normal population and the one missense scored benign by in silico criteria. To conclude, our observations suggest that AMBRA1 may function as a classical tumor suppressor gene in sporadic parathyroid adenomas at very low frequency. Further study may include protein expression analysis of AMBRA1 to investigate influences on gene expression. To further elucidate the mechanisms behind cyclin D1 overexpression in parathyroid adenomas, other molecules that participate in its degradation should be explored as potential tumor suppressors. Presentation: Saturday, June 17, 2023

Abstract 5279: Molecular analysis of AMBRA1 as a candidate tumor suppressor in sporadic parathyroid adenomas

Abstract Primary hyperparathyroidism is a common endocrine disorder that is most often caused by a sporadic single-gland parathyroid adenoma. Currently, the only known and experimentally validated oncoprotein for parathyroid adenomas is cyclin D1. Overexpression of cyclin D1 has been reported in 20-40% of cases; about 8 percent of cases harbor a DNA rearrangement involving the cyclin D1 (CCND1) locus. Other molecular mechanisms underlying this cyclin D1 overexpression have yet to be identified. Here, we explored a potential parathyroid tumorigenic mechanism that could increase cyclin D1 stability through a defect in the AMBRA1 E3 ligase adaptor, a molecule responsible for its degradation. Evidence that AMBRA1 may be a tumor suppressor gene includes: accumulation of cyclin D with hyperproliferation in AMBRA1-deficient cells, context-dependent growth of tumors in Ambra1-deficient mice, and the correlation between low levels of AMBRA1 and poor clinical outcomes in The Cancer Genome Atlas. Therefore, we examined AMBRA1 for evidence of tumor suppressor-type inactivation in a cohort of 98 typical parathyroid adenomas. Tumor genomic DNA was subjected to PCR-amplification of all 18 coding exons of AMBRA1, followed by Sanger sequencing. Sequences were analyzed by comparison to the normal reference sequence (ENST00000683756.1), seeking to assess any observed intragenic or splice-site variants as potential or likely loss-of-function mutations. Thus far, with 97 percent of the coding region for this 1298-amino acid protein fully sequenced, we have identified an inactivating mutation in 1 of 98 tumors (1%): c.126G>A, resulting in an early stop codon p.Trp42. This mutation showed loss of heterozygosity and was confirmed to be somatic by sequencing of the patient’s matched germline DNA. We also identified likely non-pathogenic variants in 5 tumors: missense variant c.3385G>T (p.Ala1129Ser), 3’ UTR variants c.*576A>T and c.*491T>C and synonymous variants c.579C>T and c.2776C>T, each in a single tumor. 4 of 5 were found as germline variants in the normal population and the one missense scored as benign by in silico criteria. To conclude, our observations suggest that AMBRA1 may function as a classical tumor suppressor gene in sporadic parathyroid adenomas at very low frequency. Further study may include protein expression analysis of AMBRA1 to investigate influences on gene expression. To further elucidate the mechanisms behind cyclin D1 overexpression in parathyroid adenomas, other molecules that participate in its degradation should be explored as potential tumor suppressors. Citation Format: Stephanie Chinwo, Justin Bellizzi, Jessica Costa-Guda, Andrew Arnold. Molecular analysis of AMBRA1 as a candidate tumor suppressor in sporadic parathyroid adenomas. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5279.

PSAT176 Molecular Analysis of AMBRA1 as a Candidate Tumor Suppressor in Sporadic Parathyroid Adenomas

Abstract Primary hyperparathyroidism is a common endocrine disorder that is most often caused by a sporadic single-gland parathyroid adenoma. Currently, the only known and experimentally validated oncoprotein for parathyroid adenomas is cyclin D1. The overexpression of cyclin D1 protein has been reported in twenty to forty percent of cases. Eight percent of this overexpression can be explained by DNA rearrangement involving the cyclin D1 locus. The remaining molecular mechanisms behind this overexpression have yet to be identified. In this study, we explored a potential parathyroid tumorigenic mechanism that could increase cyclin D1 stability through a defect in the AMBRA1 E3 ligase adaptor, a molecule responsible for its degradation. Evidence that AMBRA1 may be a tumor suppressor gene includes the accumulation of cyclin D with hyperproliferation in AMBRA1-deficient cells, the context-dependent growth of tumors in Ambra1-deficient mice, and the finding that low levels of AMBRA1 correlated with poor clinical outcomes in The Cancer Genome Atlas (TCGA). We have therefore proceeded examine AMBRA1 for evidence of tumor suppressor-type inactivation within a cohort of parathyroid adenomas. Genomic DNA from twenty-six typical sporadic parathyroid adenomas was subjected to PCR-amplification of all eighteen coding exons of AMBRA1, followed by Sanger sequencing. Sequences were analyzed by comparison to the normal reference sequence, seeking to assess any observed intragenic or splice-site variants as potential or likely loss-of-function mutations. Thus far, with ninety-seven percent of the coding region fully sequenced, no intragenic AMBRA1 inactivating mutations were observed. Three known single nucleotide polymorphisms, considered non-pathogenic, were identified: missense variant c.3028G>T, and 3' UTR variants c.*491T>C and c.*576A>T, each in a single tumor. Future study of increased numbers of parathyroid adenomas is needed to exclude the possibility that AMBRA1 inactivation could play a driver role in a small percentage of cases, and the possibility that AMBRA1 might contribute less directly e.g. via secondarily altered expression also warrants investigation. Nonetheless, our current observations strongly suggest that AMBRA1 does not have a frequent role as a classic 2-hit tumor suppressor gene in sporadic parathyroid adenomas. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m.

Histone deacetylase inhibitor abexinostat affects chromatin organization and gene transcription in normal B cells and in mantle cell lymphoma

Mantle cell lymphoma (MCL) is a rare lymphoma caused by the t(11:14) juxtaposing the cyclin D1 (CCND1) locus on chromosome 11 and the immunoglobulin heavy chain (IgH) locus on chromosome 14. Several new treatments are proposed for MCL, including histone deacetylase inhibitors (HDACi). We have studied gene expression and chromatin organization in the translocated 11q13 locus in MCL cells as compared to lymphoblastoid cell lines as well as the effect of HDACi abexinostat on chromatin organization and gene expression in the 11q13 locus. We have identified a cluster of genes overexpressed in the translocation region on chromosome 11 in MCL cells. Abexinostat provokes a genome-wide disaggregation of heterochromatin. The genes upregulated after the t(11;14) translocation react to the HDACi treatment by increasing their expression, but their gene promoters do not show significant alterations in H3K9Ac and H3K9me2 levels in abexinostat-treated cells.

Cyclin D1 and CHEK2 polymorphic variants as low risk susceptibility alleles in DTC patients

Differentiated thyroid cancer (DTC) originates from thyroid follicular epithelial cells and belongs to a group of slowly progressing tumors with relatively good prognosis. However, optimal results in the treatment of these tumors via coordinated multimodal therapy are achieved mostly by early diagnosis. A serious problem in course of DTC are recurrences and metastasis. Conversion from well differentiated thyroid cancer to aggressive anaplastic carcinoma is possible, which underlines the importance of effective diagnosis and characterisation at the molecular level as well. A genetic background of DTC is not clear. There are known several mutations detected in the thyroid tumor cells, but genetic factors predisposing to promotion of carcinogenesis of the thyroid gland are still unexplained. The CHEK2 gene (checkpoint kinase 2) is located in position 22q12.1 and spans 54.6 kb. It encodes the human ortholog of the yeast checkpoint kinases Cds1 and Rad53. In response to the damage of genetic material and inhibition of the replication process, the protein is activated by phosphorylation. Mutations in the CHEK2 gene are evidenced to be a multicancer predisposition factor. The protein product of this gene is a cell cycle checkpoint regulator, tumor suppressor and is a member of subfamily of serine/threonine protein kinases. The CCND1 gene (11q13) encodes the cyclin D1, a protein which belongs to the highly conserved cyclin family, that serve as regulators of CDK kinases. Cyclin D1 is a key regulatory protein which plays an important role in the transition from the G1 to the S phase of cell cycle during cell division. The cancer relevance of the cyclin D1 gene was apparent upon its identification in 1991. The cyclin D1 locus was initially identified based on its involvement in a chromosomal rearrangement of benign parathyroid tumors. The CCND1 gene alterations which influence cell cycle progression are frequently observed in a variety of tumors and may contribute to tumorigenesis. The aim of the study was to examine two sequence variants in CHEK2 p.R145W (c.433C>T, rs137853007 and p. I157T c.470T>C, rs17879961)and CCND1 (c.723G>A (rs9344; p.Pro241= and c.669C>T, rs3862792; p.Phe223=) genes in a group of Polish patients with differentiated thyroid cancer and control individuals from Polish population. We examined a cohort of 647 Polish patients diagnosed with differentiated thyroid carcinoma and Polish population included 799 subjects (615 females and 184 males). As a genotyping technique we used pyrosequencing. In order to determine statistical significance of differences in genotypic and allelic frequency, observed among compared groups, we used GraphPad Prism 4 (chi-square distribution and Fisher exact test). We did not observe noteworthy variability in p.R145W (c.433C>T, rs137853007), but in analysis of p.I157T sequence variant (c.470T>C, rs17879961) we demonstrated statistically significant differences in allele frequencies (p=0.005, OR=2.014, C.I.=[1.348-3.010]). In a case of CCND1 gene we observed statistically higher frequency of allele A in rs9344 locus in DTC patients (p=0.02498, OR=1.191, C.I.=[1.022-1.387]). We observed also a statistically significant difference between genotypes in subjected group. Genotype AA occurs significantly to be more frequent in patients with DTC (p=0.02023, OR=1.452, C.I.=[1.059-1.989]) than in control population group. We did not notice any statistically important differences in frequencies of genotypes or alleles in rs3862792 locus in subjected groups. * Correspondence: szymon.h@amu.edu.pl The NanoBioMedical Center, Adam Mickiewicz University, Poznan, Poland Full list of author information is available at the end of the article Hryhorowicz et al. Hereditary Cancer in Clinical Practice 2015, 13(Suppl 1):A15 http://www.hccpjournal.com/content/13/S1/A15

Retroviruses hijack chromatin loops to drive oncogene expression and highlight the chromatin architecture around proto-oncogenic loci.

The majority of the genome consists of intergenic and non-coding DNA sequences shown to play a major role in different gene regulatory networks. However, the specific potency of these distal elements as well as how these regions exert function across large genomic distances remains unclear. To address these unresolved issues, we closely examined the chromatin architecture around proto-oncogenic loci in the mouse and human genomes to demonstrate a functional role for chromatin looping in distal gene regulation. Using cell culture models, we show that tumorigenic retroviral integration sites within the mouse genome occur near existing large chromatin loops and that this chromatin architecture is maintained within the human genome as well. Significantly, as mutagenesis screens are not feasible in humans, we demonstrate a way to leverage existing screens in mice to identify disease relevant human enhancers and expose novel disease mechanisms. For instance, we characterize the epigenetic landscape upstream of the human Cyclin D1 locus to find multiple distal interactions that contribute to the complex cis-regulation of this cell cycle gene. Furthermore, we characterize a novel distal interaction upstream of the Cyclin D1 gene which provides mechanistic evidence for the abundant overexpression of Cyclin D1 occurring in multiple myeloma cells harboring a pathogenic translocation event. Through use of mapped retroviral integrations and translocation breakpoints, our studies highlight the importance of chromatin looping in oncogene expression, elucidate the epigenetic mechanisms crucial for distal cis-regulation, and in one particular instance, explain how a translocation event drives tumorigenesis through upregulation of a proto-oncogene.

Genomic high-resolution profiling of single CKpos/CD45neg flow-sorting purified circulating tumor cells from patients with metastatic breast cancer.

Circulating tumor cells (CTCs) are promising surrogate markers for systemic disease, and their molecular characterization might be relevant to guide more individualized cancer therapies. To enable fast and efficient purification of individual CTCs, we developed a work flow from CellSearch(TM) cartridges enabling high-resolution genomic profiling on the single-cell level. Single CTCs were sorted from 40 CellSearch samples from patients with metastatic breast cancer using a MoFlo XDP cell sorter. Genomes of sorted single cells were amplified using an adapter-linker PCR. Amplification products were analyzed by array-based comparative genomic hybridization, a gene-specific quantitative PCR (qPCR) assay for cyclin D1 (CCND1) locus amplification, and genomic sequencing to screen for mutations in exons 1, 9, and 20 of the phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) gene and exons 5, 7, and 8 of the tumor protein p53 (TP53) gene. One common flow-sorting protocol was appropriate for 90% of the analyzed CellSearch cartridges, and the detected CTC numbers correlated positively with those originally detected with the CellSearch system (R(2) = 0.9257). Whole genome amplification was successful in 72.9% of the sorted single CTCs. Over 95% of the cells displayed chromosomal aberrations typical for metastatic breast cancers, and amplifications at the CCND1 locus were validated by qPCR. Aberrant CTCs from 2 patients harbored mutations in exon 20 of the PIK3CA gene. This work flow enabled effective CTC isolation and provided insights into genomic alterations of CTCs in metastatic breast cancer. This approach might facilitate further molecular characterization of rare CTCs to increase understanding of their biology and as a basis for their molecular screening in the clinical setting.

Combined Epigenetic and Immunotherapy For Newly Diagnosed Mantle Cell Lymphoma: Correlative Studies Suggest The Importance Of Enhanced ADCC, Mechanisms of Resistance and Cyclin D1 Nuclear Localization Genotype

Previously, we reported that epigenetic therapy with cladribine, SAHA, and rituximab (SCR) for newly diagnosed mantle cell lymphoma was remarkably effective, with 100% overall response rate, 85-90% CR rate, and durable responses( Hasanali, AACR,2013 LBA140). Over 40 patients now have been enrolled with all patients completing therapy. Final response and CR rates will be reported at the meeting. This abstract will focus on the correlative studies performed as part of this trial.Cladribine, a purine analog with reported epigenetic activity was shown here by HELP assays to inhibit DNA methylation in vivo in 6 patients with leukemic MCL. Similar activity was also observed in two MCL and two CLL patients treated with cladribine without vorinostat off trial, suggesting cladribine is a DNA hypomethylating agent. Due to cladribine's ability to inhibit the enzyme SAH hydrolase and thus inhibit the donation of methyl groups by S-adenosyl methionine (SAM), we assayed the ability of cladibine to inhibit histone methylation in vitro by Western blot analysis and in vitro assays of histone methyltransferase (HMT) activity on H3lys9 and H3lys27. Both assays demonstrated inhibition of methylated histones (Western) and HMT activity using MCL cell cells and nuclear extract at concentrations of cladribine in the 10-20 um range, higher than the in vivo concentration of 10-20 nm. These observations could be due to the lack of sensitivity of these assays, and more sensitive assays are in development.Studies to help elucidate the mechanism of action of synergy of epigenetic drugs with the monoclonal antibody rituximab were performed. Using cells from patients with leukemic MCL treated with SCR, we assayed for characteristic changes of apoptosis using Western blotting and TUNEL assays. None were detected. We were unable to observe complement mediated cytoxicity in vitro using human serum and rituximab with added cladribine or vorinostat. With ADCC being the primary mechanism of presumed combined epigenetic and rituximab synergy, we investigated ADCC further. CD137 transcriptional upregulation was seen in several but not all treated patients, and some patients showed up regulation of perforin and granzyme mRNA by QRTPCR. An NK cell line, NKL, showed transcriptional upregulation of CD137 after treatment with cladribine and vorinostat.A polymorphism at an intron-exon junction effects the nuclear localization of cyclin D1 by removing a nuclear export signal. Although there is published evidence supporting the role of nuclear cyclin D1 in increased oncogenesis, the role of this polymorphism in MCL remains controversial. Samples from peripheral blood of patients on trial were genotyped at the cyclin D1 locus as AA, AG, or GG, with the A allele being the loss of function allele. The presence of the A allele strongly correlated with the blastic phenotype and the lack of complete remission after SCR therapy, with both being statistically significant (table 1). Immunofluorescent studies with cyclin D1 antibodies showed nuclear and cytoplasmic localization as predicted in patients with the AA and GG genotypes (Fig 1). The heterozygotes are under investigation and will be reported. [Display omitted] [Display omitted] The mechanism of resistance to SCR was studied in a patient with blastic, leukemic MCL. A 63 male achieved complete remission after two cycles of SCR. Subsequently, he developed neurologic symptoms and was found to have CNS disease. At autopsy, CD20+ disease was found in his CNS and CD20- disease was found systemically. A cell line was established from his peripheral blood that showed significantly reduced levels of CD20 mRNA. Treatment of these cells with a variety of epigenetic drugs was unable to upregulate CD20 mRNA. These cells have been in continuous culture for over 1 year and continue to show diminished levels of CD20 mRNA and protein. Epigenetic changes at the promoter are being studied by chromatin immunoprecipation (ChiP) assays. Disclosures:No relevant conflicts of interest to declare.

Biology and Treatment of Mantle Cell Lymphoma in the Genomic Era.

Abstract SCI-28The t11;14 translocation, the genetic hallmark of MCL, drives cyclin D1 expression in the tumor cells and historically facilitated the separation of MCL into a distinct entity. FISH cytogenetics are part of the workup and can be particularly helpful to separate leukemic MCL from CLL. Morphology and clinical course of MCL are heterogeneous and might have suggested the presence of different entities. Gene expression profiling answered this concern and provided several important insights: 1. despite the clinical heterogeneity, MCL has a characteristic gene expression profile supporting the accuracy of current diagnostic methods; 2. cyclin D1 negative MCL has the same diagnostic pathologic and gene expression features as cyclin D1 positive MCL, and 3. a gene expression based measure of tumor proliferation is a potent predictor of outcome and identifies patients with survival probabilities ranging from less than 1 year (highly proliferative tumors) to more than 6 years (low proliferation).1 Biologically, the gene expression based proliferation score integrates several acquired genetic changes in the tumor that include deletions of the INK4a/ARF locus encoding the tumor suppressors p14 and p16, amplification of BMI1, and secondary changes in the cyclin D1 locus. Mutations and deletions that alter the structure of the 3'UTR can enhance cyclin D1 mRNA stability,2 and the loss of miR binding sites in this region can enhance protein translation.3 These changes increase cyclin D1 protein resulting in increased proliferation. Additional genetic lesions such as deletions of ATM and p53 affect DNA damage responses pathways. The high frequency of secondary genetic changes in MCL cells may indicate genomic instability and the presence of additional chromosomal aberrations and certain genetic alterations hold prognostic information.4 With the continued refinement of whole genome genetic approaches the goal of identifying crucial pathways and possible driver genes in the pathogenesis of MCL may be within reach. MCL characterized by an antiapoptotic phenotype combined with features of aggressive lymphomas remains an incurable disease and having the worst outcome among all B-cell lymphomas. Biologic markers that predict treatment response and that could give way to targeted therapy have remained elusive. Several new drugs could help overcome treatment resistance and new analytic tools when incorporated into clinical trials may help dissect mechanisms of drug action and resistance. Our approach has been to incorporate gene expression profiling into a clinical trial of bortezomib to directly monitor the effects of the treatment on tumor biology in vivo. We identified an integrated stress response to bortezomib in sensitive tumors that may yield clinically usefully predictors of sensitivity and that could guide the development of improved therapies. 1. Rosenwald A, Wright G, Wiestner A, et al. The proliferation gene expression signature is a quantitative integrator of oncogenic events that predicts survival in mantle cell lymphoma. Cancer Cell. 2003;3:185-197. 2. Wiestner A, Tehrani M, Chiorazzi M, et al. Point mutations and genomic deletions in Cyclin D1 create stable truncated mRNAs that are associated with increased proliferation rate and shorter survival in mantle cell lymphoma. Blood. 2007. 3. Chen RW, Bemis LT, Amato CM, et al. Truncation in CCND1 mRNA alters miR-16-1 regulation in mantle cell lymphoma. Blood. 2008;112:822-829. 4. Salaverria I, Espinet B, Carrio A, et al. Multiple recurrent chromosomal breakpoints in mantle cell lymphoma revealed by a combination of molecular cytogenetic techniques. Genes Chromosomes Cancer. 2008;47:1086-1097. DisclosuresOff Label Use: Bortezomib in previously untreated patients with MCL.

Cyclin D1 fine-tunes the neurogenic output of embryonic retinal progenitor cells

BackgroundMaintaining the correct balance of proliferation versus differentiation in retinal progenitor cells (RPCs) is essential for proper development of the retina. The cell cycle regulator cyclin D1 is expressed in RPCs, and mice with a targeted null allele at the cyclin D1 locus (Ccnd1-/-) have microphthalmia and hypocellular retinas, the latter phenotype attributed to reduced RPC proliferation and increased photoreceptor cell death during the postnatal period. How cyclin D1 influences RPC behavior, especially during the embryonic period, is unclear.ResultsIn this study, we show that embryonic RPCs lacking cyclin D1 progress through the cell cycle at a slower rate and exit the cell cycle at a faster rate. Consistent with enhanced cell cycle exit, the relative proportions of cell types born in the embryonic period, such as retinal ganglion cells and photoreceptor cells, are increased. Unexpectedly, cyclin D1 deficiency decreases the proportions of other early born retinal neurons, namely horizontal cells and specific amacrine cell types. We also found that the laminar positioning of horizontal cells and other cell types is altered in the absence of cyclin D1. Genetically replacing cyclin D1 with cyclin D2 is not efficient at correcting the phenotypes due to the cyclin D1 deficiency, which suggests the D-cyclins are not fully redundant. Replacement with cyclin E or inactivation of cyclin-dependent kinase inhibitor p27Kip1 restores the balance of RPCs and retinal cell types to more normal distributions, which suggests that regulation of the retinoblastoma pathway is an important function for cyclin D1 during embryonic retinal development.ConclusionOur findings show that cyclin D1 has important roles in RPC cell cycle regulation and retinal histogenesis. The reduction in the RPC population due to a longer cell cycle time and to an enhanced rate of cell cycle exit are likely to be the primary factors driving retinal hypocellularity and altered output of precursor populations in the embryonic Ccnd1-/- retina.

Gab2-Mediated Signaling Promotes Melanoma Metastasis

Metastatic melanoma is a disease with a poor prognosis that currently lacks effective treatments. Critical biological features of metastasis include acquisition of migratory competence, growth factor independence, and invasive potential. In an attempt to identify genes that contribute to melanoma pathogenesis, a genome-wide search using bacterial artificial chromosome array comparative genomic hybridization and single nucleotide polymorphism arrays in a series of 64 metastatic melanoma samples and 20 melanoma cell lines identified increased copy numbers of Gab2 located on 11q14.1. Gab2 is an adaptor protein that potentiates the activation of the Ras-Erk and PI3K-Akt pathways and has recently been implicated in human cancer; however, its role in melanoma has not been explored. In this study, we found that Gab2 was either amplified (approximately 11%) and/or overexpressed (approximately 50%) in melanoma. Gab2 protein expression correlated with clinical melanoma progression, and higher levels of expression were seen in metastatic melanomas compared with primary melanoma and melanocytic nevi. We found that overexpression of Gab2 potentiates, whereas silencing of Gab2 reduces, migration and invasion of melanoma cells. Gab2 mediated the hyperactivation of Akt signaling in the absence of growth factors, whereas inhibition of the PI3K-Akt pathway decreased Gab2-mediated tumor cell migration and invasive potential. Gab2 overexpression resulted in enhanced tumor growth and metastatic potential in vivo. These studies demonstrate a previously undefined role for Gab2 in melanoma tumor progression and metastasis.

Retinoid Chemoprevention Trials: Cyclin D1 in the Crosshairs

Successful clinical cancer chemoprevention with retinoids was promised by compelling preclinical and early clinical evidence. Indeed, the concept of cancer chemoprevention came largely from studies showing retinoid suppression of epithelial carcinogenesis ([1][1]–[4][2]). Yet, randomized phase III

Butyrate and vitamin D3 induce transcriptional attenuation at the cyclin D1 locus in colonic carcinoma cells

In stimulating maturation of colonic carcinoma cells, the short chain fatty acid butyrate, and 1alpha,25-dihydroxyvitamin D(3), were shown to attenuate transcription of the cyclin D1 gene, giving rise to truncated transcripts of this locus. Moreover, a sequence which is highly conserved in the human, mouse, rat, and dog genome was found in the 4 kb long intron 3 of the human cyclin D1 gene, and is capable of forming a hairpin structure similar to that of microRNA precursors. The expression of this sequence is also decreased by the attenuation. Thus, the transcriptional attenuation at the cyclin D1 locus not only down-regulates the expression of this key gene in mucosal cell maturation and tumorigenesis, but may also abrogate the generation of a molecule that encompasses this conserved sequence in cyclin D1 intron 3.

Transvection mediated by the translocated cyclin D1 locus in mantle cell lymphoma

Transvection mediated by the translocated cyclin D1 locus in mantle cell lymphoma

Transvection mediated by the translocated cyclin D1 locus in mantle cell lymphoma

In mantle cell lymphoma (MCL) and some cases of multiple myeloma (MM), cyclin D1 expression is deregulated by chromosome translocations involving the immunoglobulin heavy chain (IgH) locus. To evaluate the mechanisms responsible, gene targeting was used to study long-distance gene regulation. Remarkably, these targeted cell lines lost the translocated chromosome (t(11;14)). In these MCL and MM cells, the nonrearranged cyclin D1 (CCND1) locus reverts from CpG hypomethylated to hypermethylated. Reintroduction of the translocated chromosome induced a loss of methylation at the unrearranged CCND1 locus, providing evidence of a transallelic regulatory effect. In these cell lines and primary MCL patient samples, the CCND1 loci are packaged in chromatin-containing CCCTC binding factor (CTCF) and nucleophosmin (NPM) at the nucleolus. We show that CTCF and NPM are bound at the IgH 3′ regulatory elements only in the t(11;14) MCL cell lines. Furthermore, NPM short hairpin RNA produces a specific growth arrest in these cells. Our data demonstrate transvection in human cancer and suggest a functional role for CTCF and NPM.

CTCF and Nucleophosmin Participate in Long Distance Cyclin D1 Deregulation in Mantle Cell Lymphoma.

Cyclin D1 expression in B cells is deregulated by long distance chromosome translocations involving the immunoglobulin heavy chain (IgH) locus in mantle cell lymphoma (MCL). Ge. Using chromatin immunoprecipitation (ChIP) assays, we show that the cyclin D1 (CCND1) loci in MCL cell lines and MCL patient samples are packaged in chromatin containing CCCTC binding factor (CTCF) and nucleophosmin (NPM). Using allelic chromatin immunoprecipitation (ChIP) assays, we demonstrate that both the translocated and nontranslocated cyclin D1 alleles bind both CTCF and NPM, while RNA polymerase II is only bound to the translocated allele. Sequential ChIP assays demonstrated CTCF and NPM binding at the cyclin D1 loci in MCL cells. By immunoFISH, we demonstrate that the translocated and nontranslocated cyclin D1 loci colocalize at the nucleolus. CTCF and NPM are bound at the IgH 3′ regulatory elements only in MCL cell lines. NPM shRNA effects a specific growth arrest in these cells. These data demonstrate a role for CTCF and NPM in long distance cyclin D1 deregulation in mantle cell lymphoma.

Transvection Mediated by the Translocated Cyclin D1 Gene in Mantle Cell Lymphoma.

Cyclin D1 expression in B cells is deregulated by chromosome translocations involving the immunoglobulin heavy chain (IgH) locus in mantle cell lymphoma (MCL). Gene targeting experiments produced MCL cell lines that had lost the translocated t(11;14) and no longer expressed cyclin D1. In these cyclin D1 (−) cells, the nonrearranged cyclin D1 (CCND1) locus reverts from CpG hypomethylated to hypermethylated. Reintroduction of the translocated chromosome by somatic cell hybrid fusion induces loss of methylation at the unrearranged CCND1 locus. Thus, the translocated chromosome exerts a transallelic effect on the unrearranged CCND1 locus in B cells that resembles transvection in Drosophilia. Control somatic cell fusion experiments with a nontranslocated cyclin D1 locus do not demonstrate transvection effects. We also have evidence for pairing of the translocated and nontranslocated cyclin D1 loci in MCL cell lines and MCL patient samples. This pairing is not related to DNA replication, as it is observed in flow sorted G1 cells and not in cyclin D1 expressing breast cancer cells or in B lymphocytes. In addition, to pairing of the cyclin D1 loci, we also have demonstrated translocation specific small RNAs in MCL cells upstream of the cyclin D1 gene.

Transvection Mediated by the Translocated Cyclin D1 Locus in B Cell Malignancies.

Translocations involving the immunoglobulin heavy chain gene locus (IgH) are common in B cell malignancies. One common target gene is cyclin D1, which is deregulated in most patients with mantle cell lymphoma (MCL) and 20–30% of patients with multiple myeloma (MM). The cyclin D1 locus represents a model locus to study the role of epigenetics in B cell malignancy. Cyclin D1 is known not to be expressed in normal lymphocytes, where the locus is DNA hypomethylated. In genetic variants that have lost the translocated chromosome, the cyclin D1 locus assumes a densely methylated configuration. Reintroduction of the translocated chromosome by somatic cell fusion reverts the methylated normal locus to a hypomethylated state in trans. Thus, the translocated chromosome exerts a trans DNA hypomethylating effect on the normal chromosome that resembles transvection in Drosophila. This is the first evidence of transvection in a human malignancy. Similar to transvection in Drosophila and paramutation in plants, evidence for pairing of the cyclin D1 loci and the participation of small RNA's in this process has been obtained. Combined FISH/ immunofluorescent studies have shown the colocalization of both the normal and translocated chromosome 11's at the nucleolus. Using chromatin immunoprecipitation assays, we show that CTCF and nucleophosmin are present at both the normal and translocated chromosomes in B cells expressing cyclin D1 but not in B lymphocytes. Tethering of the translocated and nontranslocated cyclin D1 loci by CTCF/nucleophosmin at the nucleolus provides physical and spatial juxtaposition of the cyclin D1 loci permissive for transvection.

Amplification of the cyclin D1 locus 11q13 in melanoma metastases: association to cyclin D1 protein expression

Karolinska Institute, Huddinge, Sweden Abstracts of the Perspectives in Melanoma X and The Third Annual International Melanoma Research Congress, 14–16 September 2006, Noordwijk, The Netherlands

Gain of 11q/cyclin D1 overexpression is an essential early step in skin cancer development and causes abnormal tissue organization and differentiation

Non-melanoma skin cancers, in particular keratoacanthomas (KAs) and squamous cell carcinomas (SCCs), have become highly frequent tumor types especially in immune-suppressed transplant patients. Nevertheless, little is known about essential genetic changes. As a paradigm of 'early' changes, that is, changes still compatible with tumor regression, we studied KAs by comparative genomic hybridization and show that gain of chromosome 11q is not only one of the most frequent aberration (8/18), but in four tumors also the only aberration. Furthermore, 11q gain correlated with amplification of the cyclin D1 locus (10/14), as determined by fluorescence in situ hybridization, and overexpression of cyclin D1 protein (25/31), as detected by immunohistochemistry. For unraveling the functional consequence, we overexpressed cyclin D1 in HaCaT skin keratinocytes. These cells only gained little growth advantage in conventional and in organotypic co-cultures. However, although the control vector-transfected cells formed a well-stratified and orderly differentiated epidermis-like epithelium, they showed deregulation of tissue architecture with an altered localization of proliferation and impaired differentiation. The most severe phenotype was seen in a clone that additionally upregulated cdk4 and p21. These cells lacked terminal differentiation, exhibited a more autonomous growth in vitro and in vivo and even formed tumors in two injection sites with a growth pattern resembling that of human KAs. Thus, our results identify 11q13 gain/cyclin D1 overexpression as an important step in KA formation and point to a function that exceeds its known role in proliferation by disrupting tissue organization and thereby allowing abnormal growth.