Cyclin D1a Research Articles

Expression Of Cyclin D1 Protein Isoforms And Its Prognostic Significance In Cervical Cancer.

IntroductionCyclin D1 had been associated with different clinical and pathological stages of cervical cancer; however, few studies had focused on its correlation with cervical cancer prognosis. Therefore, this study aimed to assess the expression of cyclin D1a and D1b in normal tissue, cervical cancer and cervical intraepithelial neoplasia and their effect on prognosis.MethodsExpression of cyclin D1a and D1b was detected by immunohistochemical staining in 78 cases of primary cervical cancer, 40 cases of cervical intraepithelial neoplasia, and 40 cases of normal cervical tissue.ResultsNo significant difference was observed in the expression of cyclin D1a between normal and cervical cancer tissues (P = 0.201); however, its expression was significantly higher in cervical cancer than in cervical intraepithelial neoplasia tissues (P = 0.000). Expression of cyclin D1b was higher in normal tissues than in cervical cancer tissues (P = 0.000). No significant difference was observed in the expression of cyclin D1a in cervical cancer tissues with respect to age, pathological type, clinical-stage, depth of tumor invasion, or presence of lymph node metastases (P = 0.111,0.119,0.539,0.084,0.539). COX survival analysis showed that lymph node metastasis might be an independent factor affecting postoperative recurrence (hazard risk [HR] = 0.240; 95% confidence interval [CI] = 0.968–30.156; P = 0.034).DiscussionCyclin D1a expression was associated with tumor tissue size and degree of differentiation. The expression of cyclin D1b in cervical cancer was associated with the presence of lymph node metastases. Cyclin D1a and D1b expression in cervical cancer tissue was significantly correlated. Cox survival analysis showed that the presence of lymph node metastases might serve as an independent factor affecting postoperative recurrence. The expression of cyclin D1a and D1b was not associated with cervical cancer prognosis.ConclusionAssessment of cyclin D1a and D1b expression in cervical cancer and cervical intraepithelial neoplasia revealed that cyclin D1 could not be used as a reference to assess cervical cancer patient prognosis.

An analysis of multiple biomarkers to better predict prostate cancer metastasis and death after radical prostatectomy.

54 Background: Identification of potentially lethal disease at the time of diagnosis with localized prostate cancer (PCa) remains a significant clinical issue despite a plethora of candidate biomarkers. This study evaluates a range of biomarkers previously associated with biochemical relapse (BR) in localized PCa to determine whether a combined expression model can improve detection of clinically significant cases. Methods: The Australian PCa Research Centre NSW has completed 23 studies of molecular biomarkers associated with BR in a well-described localized PCa cohort (n=324, median followup 16 years). 12 studies were excluded due to missing data. Each biomarker was analyzed as a marker for metastatic-free survival (MFS) and prostate cancer specific survival (PCSS) and then used to develop a prognostic model for clinical outcomes incorporating clinico-pathological factors. This model is currently being validated in an independent cohort. Results: The PCa cohort experienced 39 metastatic relapses (12%) and 23 PCa deaths (7%). Of 12 biomarkers (AR, AZPG1, C0S, Cyclin D1a, Cyclin D1b, E6AP, H3K18Ac, H3K4me2, Ki67, p53, PML, SGTA) assessed, only AZGP1 and Ki67 were associated with MFS (HR 2.9, 95% CI, 1.4-5.6; P=0.002, and HR 1.2, 95% CI, 1.0-1.4; P=0.03, respectively) and PCSS (HR 4.2, 95% CI, 1.7-10.5; P=0.002; and HR 1.2, 95% CI, 1.0-1.5; P=0.04, respectively). The combined panel of AZGP1 and Ki67 was an independent predictor of MFS (HR 1.9, 95% CI, 1.1-3.2; P=0.01), and PCSS (HR 3.3, 95% CI, 1.5-7.3; P=0.002) when modeled with known clinicopathological variables. The panel was more robust in predicting MFS and PCSS compared to the individual biomarkers alone and superior to other prognostic models (See table). Data from the validation cohort will be available for the meeting. Conclusions: Our novel signature of AZPG1 and Ki67 improves existing prognostication tools in predicting PCa metastasis and death. [Table: see text]

Anti-oncogenic activities of cyclin D1b siRNA on human bladder cancer cells via induction of apoptosis and suppression of cancer cell stemness and invasiveness.

The human cyclinD1 gene generates two major isoforms, cyclinD1a and cyclinD1b, by alternative splicing. Although cyclin D1b mRNA is hardly expressed in normal human tissues, it is detected in approximately 60% of human bladder cancer tissues and cell lines. In the present study, to assess the therapeutic ability of cyclinD1b siRNA, we investigated the anti-oncogenic effects of cyclinD1b siRNA on human bladder cancer cell lines, SBT31A and T24, which express cyclinD1b mRNA. Knockdown of cyclinD1b by specific siRNA significantly suppressed cell proliferation, invitro cell invasiveness and three-dimensional (3D) spheroid formation in these cell lines. Cell cycle analyses revealed that cyclinD1b siRNA inhibited G1-S transition in T24 cells. The increase in the sub-G1 fraction, morphological aberrant nuclei with nuclear fragmentation and caspase-3 activity in SBA31A cells treated with cyclinD1b siRNA showed that cyclinD1b siRNA induced apoptosis. In T24 cells, knockdown of cyclinD1b suppressed the expression of the stem cell marker CD44. Knockdown of cyclinD1b or CD44 suppressed the invasiveness under 3D spheroid culture conditions and expression of N-cadherin. Tumor growth of SBT31A cells in nude mice was significantly inhibited by cyclinD1b siRNA. Taken together, these results indicate that knockdown of cyclinD1b suppresses the malignant phenotypes of human bladder cancer cells via induction of apoptosis and suppression of cancer cell stemness and epithelial-mesenchymal transition. Applying cyclinD1b siRNA will be a novel therapy for cyclinD1b-expressing bladder cancers.

The Results of a Phase I Study using Velcade (Bortezomib), Cladribine, and Rituximab (VCR) in treating Mantle Cell Lymphoma

Background: Mantle cell lymphoma (MCL) is a moderately aggressive and incurable small to medium size B cell lymphoma. There is no standardized treatment for this disease. The conventional chemotherapy results in a high incidence of treatment related toxicity with frequent disease relapse. Cladribine is a hypomethylating agent that indirectly downregulate DNA methylation to suppress tumorigenesis. Combination of Cladribine and Rituximab showed a synergetic effect in treating B cell lymphomas. Velcade (Bortezomib) is a FDA approved proteasome inhibitor to treat relapsed/refractory MCL. In this phase 1 study, we evaluated the safety and efficiency of Valcade, Cladribine, and Rituximab (VCR) combination treatment in newly diagnosed and relapsed/refractory MCL.Patients and Methods: This is a single arm, open label, investigator initiated Phase 1 study conducted at PennState Hershey Cancer Institute. This study employed a standard 3+3 dose-escalation scheme designed to determine the maximum tolerated dose (MTD) of Cladribine in VCR regimen. The treatment scheme and Cladribine dose escalation levels (1, 2, and 3) are as follows: the therapy consisted of 6 28-day cycles. At first cycle of the treatment, Rituximab 375 mg/m2 infusion started on day 5 of the first week and then given weekly for 3 weeks; in the next 5 cycles Rituximab was given on day 5 of each cycle, and then every 2 months as the maintenance therapy. Cladribine 3-5 mg/m2 (3 mg/m2 for level 1, 4 mg/m2 for level 2, and 5 mg/m2 for level 3) infusion was given on days 1-5 for 6 cycles. If the patient's was older than 70 years old, Cladribine was only given on days 1-3 of each cycle. Velcade 1.6 mg/m2 subcutaneous injection was given on days 12, 19 and 26 for cycles 1-3, then Day 5 and 19 during Cycles 4-6, then once per month as maintenance therapy until toxicity or progression of the disease. The primary endpoint of this study was to investigate the dose-limiting toxicity (DLT), safety, and efficiency of this regimen in patients with MCL. The secondary endpoints included remission rate (RR), progression-free survival (PFS) and overall survival (OS). The analysis was done by intent to treat. This study is registered with clinicaltrials.gov (NCT01439750).Results: No subject experienced dose limited toxicity (DLT) at either level 1 or 2, one possible DLT (infectious colitis) was observed on a subject during 2nd cycle at level 3. Then no additional DLTs were seen in 3 subjects added to level 3. Overall, this study recruited 13 subjects, with a median age of 64 years old (range from 55 to 83), and a total of 11 male and 2 females. Ten subjects were never previously treated, while 3 either relapsed or failed previous treatments. The majority of subjects tolerated this regimen well. The patient with a history of DLT became the only mortality of this cohort 7 months later as a result of multiple organ failure. The overall remission (OR) rate was 85% (11/13) and complete remission (CR) rate was 62% (8/13). In newly diagnosed subject cohort, the RR and OS rates were 100% (9/9), PFR was 89% (8/9), and CR rate was 78% (7/9), of which 2 patients have been followed for more than 3 years and 4 patients for more than 2 years. However, in relapsed/refractory subject cohort, 1 subject achieved CR for 7 months but then relapsed, 2 subjects had no response. There were 2 blastoid subjects, one was newly diagnosed and achieved CR.No severe systemic toxicity was observed in this study. Bone marrow suppression caused prolonged anemia and thrombocytopenia were the most common toxicity (3/13 with ≤grade 2). Low grade peripheral neuropathies (≤grade 2) were observed in 15% (2/13). Mild fatigue was a frequent complaint.Correlative studies measured cyclin D1 expression levels using mononuclear cells as opposed to formalin-preserved tissue, a technique which allows for monitoring the levels over time. Although limited by a small sample size, the results indicated that the G/G (or A/A) genotype at G870A polymorphism site as well as a rapid decline of cyclin D1 and D1a during treatment may be correlated to a less aggressive disease course. Further studies are needed to explore the underlining mechanism.Conclusions: The VCR combination is a well tolerated, low toxicity and effective regimen for MCL, especially for untreated MCL. Cladribine 5 mg/m2 is a tolerable dose with manageable toxicity. A phase II trial to further evaluate the activity and toxicity of VCR regimen is warranted. DisclosuresNo relevant conflicts of interest to declare.

Akt-dependent activation of Erk by cyclin D1b contributes to cell invasiveness and tumorigenicity.

A total of two major isoforms, cyclin D1a and cyclin D1b, are generated from the human cyclin D1 gene by alternative splicing. Cyclin D1b is scarcely expressed in normal tissues; however, it is expressed at a high frequency in certain types of cancerous tissue. The present authors previously constructed cyclin D1b transgenic (Tg) mice and identified rectal tumors, including adenocarcinoma and sessile serrated adenoma, in 62.5% of female Tg mice. In addition, the present authors indicated that cyclin D1b expression enhances phosphorylation of extracellular signal-regulated kinase (Erk) in these rectal tumors, and in mouse embryonic fibroblast (MEF) cells and human 293T cells. In the present study, it was initially demonstrated that cyclin D1b has the ability to enhance cell invasiveness by itself; it additionally increases cell invasiveness, anchorage-independent growth and tumorigenicity in cooperation with an activated K-ras oncogene in MEF cells. Phosphorylation of Akt was increased in cyclin D1b-expressing MEF cells and in the rectal tumor tissues of cyclin D1b Tg mice. Phosphorylation of Akt was also enhanced by transfection of cyclin D1b, but not cyclin D1a, in human 293T cells. Treatment with an Akt inhibitor suppressed phosphorylation of Erk in 293T cells expressing cyclin D1b and D1bTgRT cells established from rectal cancer of the cyclin D1b Tg mouse. Furthermore, the Akt inhibitor suppressed the invasiveness of D1bTgRT cells and the tumor growth of these cells in nude mice when the Akt inhibitor was injected into the tumors. These results indicate that cyclin D1b activates Erk through Akt, and that activation of Akt contributes to the tumorigenicity of the cyclin D1b Tg mice. Inhibitors targeting the phosphoinositide 3-kinase/Akt signaling pathway are thus expected to have therapeutic potential in a variety of human cancer types expressing cyclin D1b.

Abstract 460: Diagnostic and prognostic value of expression of CCND1 splice variant cyclin D1b in papillary thyroid carcinoma

Abstract Background: An active cyclin D1 isoform, cyclin D1b, arises as a result of a failure to splice the fourth intron of the CCND1 pre-mRNA transcript. The expression of cyclin D1b has been associated with tumorigenesis and tumor progression in many types of human cancer. However, no studies with cyclin D1b have yet been reported in the thyroid cancer. Methods: We investigated the CCND1 rs9344 (cDNA 870) polymorphism by TaqMan SNP genotyping assay and expression profiles of mRNA and protein of full-length cyclin D1 (cyclin D1a) and cyclin D1b by quantative real-time PCR and immunohistochemistry in 286 thyroid tumors including 16 nodular hyperplasias, 33 follicular adenomas (FA), 179 papillary thyroid carcinomas (PTC), 32 follicular thyroid carcinomas (FTC), 5 poorly differentiated carcinomas (PDC), 4 anaplastic thyroid carcinomas (ATC), and 17 medullary thyroid carcinomas (MTC). The relationships between cyclin D1 expression levels and clinicopathological features were analyzed. Results: The cyclinD1b mRNA expression level in all thyroid tumors was significantly higher in the AA and GA genotype groups compared with the GG genotype group (p<0.001 and p = 0.006, respectively). The mRNA expression levels of cyclin D1b was significantly higher in PTCs when compared to benign or other malignant tumors (p<0.001) while no significant difference in cyclin D1a mRNA expression was observed according to the genotypes or tumor types. PTCs showed similar expression levels of cyclin D1b mRNA regardless of histologic subtypes, even when compared with follicular variant. In immunohistochemistry analysis, cyclin D1b overexpression was observed in PTC, PDC, ATC, and MTC, but not in FA and FTC. Cytoplasmic overexpression of cyclin D1b was significantly associated with advanced tumor stage in all PTCs (p = 0.025). Nuclear overexpression of cyclin D1b was associated with an increased incidence of lymph node metastases (p = 0.014) in classic PTCs. Conclusion: The A allele of the rs9344 polymorphism predisposes for cyclin D1b production in thyroid cancer. Cyclin D1b overexpression has a diagnostic biomarker utility to differentiate PTC from benign follicular nodules and may be functionally involved in the progression of the disease. Citation Format: Chan Kwon Jung, Sora Jeon, Yourha Kim, Ahwon Lee, Gyeong Sin Park. Diagnostic and prognostic value of expression of CCND1 splice variant cyclin D1b in papillary thyroid carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 460.

MicroRNA-195 inhibits proliferation of cervical cancer cells by targeting cyclin D1a.

Cervical cancer is one of the most frequent gynecological malignancies in women worldwide. MicroRNA-195 (miR-195) was recently found highly expressed in cervical cancer. However, the role of miR-195 in the pathology of cervical cancer remains poorly understood. In this study, we first confirmed the downregulation of miR-195 in primary cervical cancer tissues. For the functional study, we introduced the sequences of miR-195 or miR-195 inhibitor into Hela and SiHa cervical cancer cell lines. Overexpression of miR-195 inhibited the proliferation of both Hela and SiHa cells. In contrast, reducing the endogenous miR-195 level by miR-195 inhibitor promoted the proliferation of cervical cancer cells. Flow cytometric assay showed that overexpression of miR-195 induced G1 phase arrest, whereas miR-195 inhibitor shortened G1 phase of cervical cancer cells. In addition, the suppressive role of miR-195 in cell cycle was also demonstrated by the western blot results of various cell cycle indicators, such as phosphorylated retinoblastoma (p-Rb) and proliferating cell nuclear antigen (PCNA), in the gain and loss of function experiments. Furthermore, Dual-Luciferase Reporter Assay revealed that miR-195 targeted the 3'-untranslated region of cyclin D1a transcript, such as to regulate cyclin D1 expression. In summary, our results suggest that miR-195 acts as a suppressor in the proliferation and cell cycle of cervical cancer cells by directly targeting cyclin D1a mRNA.

Influence of CCND1 G870A polymorphism on the risk of HBV-related HCC and cyclin D1 splicing variant expression in Chinese population

The G870A polymorphism in the exon 4/intron 4 boundary of CCND1 gene is thought to influence the generation of two mRNAs (cyclin D1a and cyclin D1b). The “A” allele codes for a truncated variant, cyclin D1b, which may have higher transforming activity. Herein, the tumor relevance of G870A polymorphism, the association between cyclin D1 variant expression and G870A genotype, and the oncogenic potential of cyclin D1 variants in HBV-related hepatocellular carcinoma (HCC) were examined. We found that there is no significant difference of G870A distribution among the HCC, chronic HBV (CHB) infection, cirrhotic CHB, and healthy control groups. Stratification analysis revealed that in younger patients (ages ≤ 50), cirrhotic CHB patients with AA genotype had an increased risk of developing HCC with odds ratio of 1.943 (95 % CI 1.022–3.694, p = 0.0411) as compared with AG/GG genotypes. The two variants were both transcripted from “A” and “G” alleles, and neither cyclin D1a nor D1b production was influenced by G870A genotype in HCC. The expression of both cyclins D1a and D1b decreased in HCC tissues (p = 0.003, p = 0.005), while increased in adjacent nontumor tissues as compared with normal liver tissues (p = 0.045, p = 0.034). Overexpression of cyclin D1a or D1b could promote the cell proliferation and cell-cycle progression in Huh-7 and LO2 cell lines. Collectively, our data suggest that G870A polymorphism has only very limited predictive value for HBV-related HCC. Both cyclins D1a and D1b could promote cell proliferation, which might contribute to the potential oncogenic role of cyclin D1 variants during the precancerous cirrhotic stage of hepatocarcinogenesis.Electronic supplementary materialThe online version of this article (doi:10.1007/s13277-015-3401-7) contains supplementary material, which is available to authorized users.

Cyclin D1b splice variant promotes αvβ3-mediated adhesion and invasive migration of breast cancer cells

Cyclin D1b, a splice variant of the cell cycle regulator cyclin D1, holds oncogenic functions in human cancer. However, the mechanisms underlying cyclin D1b function remain poorly understood. Here we introduced wild-type cyclin D1a or cyclin D1b variant into non-metastatic MCF-7 cells. Our results show that ectopic expression of cyclin D1b promotes invasiveness of the cancer cells in a cyclin D1a independent manner. Specifically, cyclin D1b is found to modulate the expression of αvβ3, which characterizes the metastatic phenotype, and enhance tumor cell invasive potential in cooperating with HoxD3. Notably, cyclin D1b promotes αvβ3-mediated adhesion and invasive migration, which are associated with invasive potential of breast cancer cells. Further exploration indicates that cyclin D1b makes breast cancer cells more sensitive to toll-like receptor 4 ligand released from damaged tumor cells. These findings reveal a role of cyclin D1b as a possible mediator of αvβ3 transcription to promote tumor metastasis.

Cyclin D1 induction of Dicer governs microRNA processing and expression in breast cancer

Cyclin D1 encodes the regulatory subunit of a holoenzyme that phosphorylates the pRB protein and promotes G1/S cell cycle progression and oncogenesis. Dicer is a central regulator of miRNA maturation, encoding an enzyme that cleaves double strand RNA or stem-loop-stem RNA into 20–25 nucleotide long small RNA, governing sequence specific gene silencing and heterochromatin methylation. The mechanism by which the cell cycle directly controls the non-coding genome is poorly understood. Here we show that cyclin D1−/− cells are defective in pre-miRNA processing which is restored by cyclin D1a rescue. Cyclin D1 induces Dicer expression in vitro and in vivo. Dicer is transcriptionally targeted by cyclin D1, via a cdk-independent mechanism. Cyclin D1 and Dicer expression significantly correlates in luminal A and basal-like subtypes of human breast cancer. Cyclin D1 and Dicer maintain heterochromatic histone modification (Tri-m-H3K9). Cyclin D1-mediated cellular proliferation and migration is Dicer-dependent. We conclude that cyclin D1 induction of Dicer coordinates microRNA biogenesis.

Female-specific rectal carcinogenesis in cyclin D1b transgenic mice

Human cyclin D1 generates two major isoforms via alternative splicing: cyclin D1a and cyclin D1b. Cyclin D1b is hardly expressed in normal tissues but is frequently expressed in certain types of cancer tissues. To clarify the oncogenic potential of cyclin D1b variant, we developed cyclin D1b transgenic (Tg) mice and analyzed their phenotypes. We detected rectal tumors in 63% (15/24) of the female Tg mice. All rectal tumors had the histological characteristics similar to human sessile serrated adenoma/polyps (SSA/Ps). Adenocarcinomas were also found in 53% (8/15) of the rectal tumors, suggesting that these adenocarcinomas originated from the SSA/P-like lesions. No rectal tumors were found in the ovariectomized female cyclin D1b Tg mice (0/10), indicating that ovarian hormones played a critical role in rectal carcinogenesis in these Tg mice. Both phosphorylation of Erk, without activating MEK, and expression of estrogen receptor β were elevated in the rectal tumors of female cyclin D1b Tg mice compared with normal rectums of female wild-type mice. In addition, we established a cell line, D1bTgRT, derived from a rectal cancer of female Tg mouse. Small interfering RNA-induced cyclin D1b knockdown in this cell line suppressed Erk phosphorylation, anchorage-independent growth, cell invasiveness and tumorigenicity in nude mice. In humans, expression of cyclin D1b messenger RNA was detected in 17% (1/6) of colorectal cancer cell lines and 9.7% (3/31) of colorectal cancer tissues. Taken together, these results indicate that cyclin D1b expression contributes to the female- specific rectal carcinogenesis in mouse model.

Expression of cyclin D1a and D1b as predictive factors for treatment response in colorectal cancer

Background:The aim of this study was to investigate the value of the cyclin D1 isoforms D1a and D1b as prognostic factors and their relevance as predictors of response to adjuvant chemotherapy with 5-fluorouracil and levamisole (5-FU/LEV) in colorectal cancer (CRC).Methods:Protein expression of nuclear cyclin D1a and D1b was assessed by immunohistochemistry in 335 CRC patients treated with surgery alone or with adjuvant therapy using 5-FU/LEV. The prognostic and predictive value of these two molecular markers and clinicopathological factors were evaluated statistically in univariate and multivariate survival analyses.Results:Neither cyclin D1a nor D1b showed any prognostic value in CRC or colon cancer patients. However, high cyclin D1a predicted benefit from adjuvant therapy measured in 5-year relapse-free survival (RFS) and CRC-specific survival (CSS) compared to surgery alone in colon cancer (P=0.012 and P=0.038, respectively) and especially in colon cancer stage III patients (P=0.005 and P=0.019, respectively) in univariate analyses. An interaction between treatment group and cyclin D1a could be shown for RFS (P=0.004) and CSS (P=0.025) in multivariate analysis.Conclusion:Our study identifies high cyclin D1a protein expression as a positive predictive factor for the benefit of adjuvant 5-FU/LEV treatment in colon cancer, particularly in stage III colon cancer.

Impaired nuclear export of tumor-derived c-terminal truncated cyclin D1 mutant in ESCC cancer

Cyclin D1 is a significant regulator of the G1- to S-phase transition and is often aberrant in human tumors of various origins. Although cancer-derived cyclin D1 mutants are potent oncogenes in vitro and in vivo, the mechanisms by which they contribute to neoplasia remaind to be elucidated. We previously identified a cyclin D1 mutation (Δ266-295) in esophageal cancer with deleted codons from 266 to 295 of wild-type cyclin D1, the critical COOH-terminal regulatory sequences necessary for cyclin D1 nuclear export. In the present study, this cancer-derived cyclin D1-Δ266-295 was shown to be a constitutively nuclear cyclin D1 protein with a significantly increased oncogenic potential. Moreover, the cancer-derived cyclin D1-Δ266-295 mutant was found to retain its ability to bind to and activate CDK4, which in turn phosphorylates and inactivates the pRb protein and promotes cell cycle progression. In comparison to wild-type cyclin D1a, D1-Δ266-295 exhibited enforced nuclear accumulation. In addition, the transient transfection and ectopic expression of this nuclear localized D1-Δ266-295 up-regulated endogenous Notch1 expression, indicating that the mutant retained its ability as a transcriptional regulator. Furthermore, data from the flow cytometry assay showed that D1-Δ266-295 fractionally increased >4N cell accumulation, and further analysis suggested the retriggering of DNA replication relevant to its inhibition of Cdt1 proteolysis. Therefore, the inappropriate nuclear localization of this cyclin D1 mutant may interfere with DNA replication in cultured cells, thereby contributing to genomic instability.

PACSIN 2 represses cellular migration through direct association with cyclin D1 but not its alternate splice form cyclin D1b

Cyclin D1 overexpression is a common feature of many human malignancies. Genomic deletion analysis has demonstrated a key role for cyclin D1 in cellular proliferation, angiogenesis, and cellular migration. To investigate the mechanisms contributing to cyclin D1 functions, we purified cyclin D1a-associated complexes by affinity chromatography and identified the PACSIN 2 (protein kinase C and casein kinase substrate in neurons 2) protein by mass spectrometry. The PACSIN 2, but not the related PACSIN 1 and 3, directly bound wild-type cyclin D1 (cyclin D1a) at the carboxyl terminus, and failed to bind cyclin D1b, the alternative splicing variant of cyclin D1. PACSIN 2 knockdown induced cellular migration and reduced cell spreading in LNCaP cells expressing cyclin D1a. In cyclin D1-/- mouse embryonic fibroblasts (MEFs), cyclin D1a, but not cyclin D1b, reduced the cell spreading to a polarized morphology. siPACSIN 2 had no effect on cellular migration of cyclin D1-/- MEFs. Cyclin D1a restored the migratory ability of cyclin D1-/- MEFs, which was further enhanced by knocking down PACSIN 2 with siRNA. The cyclin D1-associated protein, PACSIN 2, regulates cell spreading and migration, which are dependent on cyclin D1 expression.

Alternative Cyclin D1 Splice Forms Differentially Regulate the DNA Damage Response

The DNA damage response (DDR) activates downstream pathways including cell cycle checkpoints. The cyclin D1 gene is overexpressed or amplified in many human cancers and is required for gastrointestinal, breast, and skin tumors in murine models. A common polymorphism in the human cyclin D1 gene is alternatively spliced, resulting in cyclin D1a and D1b proteins that differ in their carboxyl terminus. Cyclin D1 overexpression enhances DNA damage-induced apoptosis. The role of cyclin D1 and the alternative splice form in regulating the DDR is not well understood. Herein cyclin D1a overexpression enhanced the DDR as characterized by induction of γH2AX phosphorylation, the assembly of DNA repair foci, specific recruitment of DNA repair factors to chromatin, and G(2)-M arrest. Cyclin D1 deletion in fibroblasts or small interfering RNA-mediated reduction of endogenous cyclin D1 in colon cancer cells reduced the 5-fluorouracil-mediated DDR. Mechanistic studies showed that cyclin D1a, like DNA repair factors, elicited the DDR when stably associated with chromatin.

Identification of ASF/SF2 as a Critical, Allele-Specific Effector of the Cyclin D1b Oncogene

The cyclin D1b oncogene arises from alternative splicing of the CCND1 transcript, and harbors markedly enhanced oncogenic functions not shared by full-length cyclin D1 (cyclin D1a). Recent studies showed that cyclin D1b is selectively induced in a subset of tissues as a function of tumorigenesis; however, the underlying mechanism(s) that control tumor-specific cyclin D1b induction remain unsolved. Here, we identify the RNA-binding protein ASF/SF2 as a critical, allele-specific, disease-relevant effector of cyclin D1b production. Initially, it was observed that SF2 associates with cyclin D1b mRNA (transcript-b) in minigene analyses and with endogenous transcript in prostate cancer (PCa) cells. SF2 association was altered by the CCND1 G/A870 polymorphism, which resides in the splice donor site controlling transcript-b production. This finding was significant, as the A870 allele promotes cyclin D1b in benign prostate tissue, but in primary PCa, cyclin D1b production is independent of A870 status. Data herein provide a basis for this disparity, as tumor-associated induction of SF2 predominantly results in binding to and accumulation of G870-derived transcript-b. Finally, the relevance of SF2 function was established, as SF2 strongly correlated with cyclin D1b (but not cyclin D1a) in human PCa. Together, these studies identify a novel mechanism by which cyclin D1b is induced in cancer, and reveal significant evidence of a factor that cooperates with a risk-associated polymorphism to alter cyclin D1 isoform production. Identification of SF2 as a disease-relevant effector of cyclin D1b provides a basis for future studies designed to suppress the oncogenic alternative splicing event.

Cyclin K and cyclin D1b are oncogenic in myeloma cells

BackgroundAberrant expression of cyclin D1 is a common feature in multiple myeloma (MM) and always associated with mantle cell lymphoma (MCL). CCND1 gene is alternatively spliced to produce two cyclin D1 mRNA isoforms which are translated in two proteins: cyclin D1a and cyclin D1b. Both isoforms are present in MM cell lines and primary cells but their relative role in the tumorigenic process is still elusive.ResultsTo test the tumorigenic potential of cyclin D1b in vivo, we generated cell clones derived from the non-CCND1 expressing MM LP-1 cell line, synthesizing either cyclin D1b or cyclin K, a structural homolog and viral oncogenic form of cyclin D1a. Immunocompromised mice injected s.c. with LP-1K or LP-1D1b cells develop tumors at the site of injection. Genome-wide analysis of LP-1-derived cells indicated that several cellular processes were altered by cyclin D1b and/or cyclin K expression such as cell metabolism, signal transduction, regulation of transcription and translation. Importantly, cyclin K and cyclin D1b have no major action on cell cycle or apoptosis regulatory genes. Moreover, they impact differently cell functions. Cyclin K-expressing cells have lost their migration properties and display enhanced clonogenic capacities. Cyclin D1b promotes tumorigenesis through the stimulation of angiogenesis.ConclusionsOur study indicates that cyclin D1b participates into MM pathogenesis via previously unrevealed actions.

Abstract 3878: Perturbations in the AR-cyclin D1 axis controls lethal tumor phenotypes in prostate cancer

Abstract Androgen receptor (AR) signaling is critical to prostate cancer development. It has been well established that AR activation promotes lethal tumor phenotypes including proliferation, survival, invasion, and therapeutic resistance. However, mechanisms by which AR promotes these phenotypes are poorly understood. Previous reports assessing cellular proliferation revealed that the AR-cyclin D1a axis plays a significant role in controlling the response to androgen. While activated AR induces cyclin D1a through mTOR mediated mechanisms, accumulated cyclin D1a serves a negative feedback control function, through the ability of the cyclin to bind and suppress further AR signaling. Importantly, this process is frequently abrogated in human prostate cancer through an alternative splicing event that results in production of cyclin D1b. Cyclin D1b is deficient in its ability to regulate AR, and has been shown in other model systems to harbor significant oncogenic activity. Importantly, we have demonstrated that nearly 1/3 of a large cohort of primary prostate cancer tumors exclusively express cyclin D1b. Thus, it is imperative to determine the molecular and cellular consequence of the alternative splicing event in prostate cancer. Endogenous D-cyclins were eliminated using hormone ablation; subsequent viral-mediated reintroduction of cyclins D1a or D1b allowed for unbiased comparison of cyclin function in the presence and absence of androgen. Gene expression profiling confirmed that D1b is unable to manage AR activity. Concordantly, alteration of androgen-sensitive genes was observed, in particular those associated with migration and invasion. In vitro manipulation of the D1 splicing event confirmed these results. Subsequent modeling of cyclin D1b function revealed that cyclin D1b confers a specific anchorage independence and migration phenotype to AR-positive prostate cancer cells. Thus, these data provide the first evidence that perturbation in the AR-cyclin D1 axis controls lethal tumor phenotypes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3878.

Cyclin D1 G870A polymorphism is associated with an increased risk of hepatocellular carcinoma in the Turkish population: Case–control study

Background: A common G to A polymorphism (G870A) in the splice donor region of exon 4 of cyclin D1 (CCND1) gene generates two mRNAs (cyclin D1a and D1b) through an alternative splicing at the site of this polymorphism. Cyclin D1a and b proteins differ in their COOH-terminus, a region involved in protein degradation. We examined the association between this CCDN1 genotype and the susceptibility to hepatocellular carcinoma (HCC) in a Turkish population. Methods: The genotype frequency of this polymorphism was determined by using a polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) assay. Hospital-based case–control study was designed consisting of 160 diagnosis subjects with hepatocellular carcinoma and 160 cancer-free control subjects matched on age, gender, smoking and alcohol status. Results: The allele frequencies of case subjects (A, 0.55; G, 0.45) were significantly different from those of control subjects (A, 0.42; G, 0.58) ( p = 0.002). The odds ratios (ORs) for the CCND1 870 GA and AA genotypes when compared with the GG genotypes were 1.39 (95% confidence interval [CI] 0.82–2.36, p = 0.22) and 2.52 (95% CI 1.38–4.62, p = 0.003) respectively. The presence of at least one CCND1 870A allele was associated with increased risk for HCC (OR, 1.73; 95% CI, 1.06–2.82, p = 0.03). When combining the GG and GA genotypes as a reference genotype, we found that the OR for the AA genotype was 2.06 (95% CI 1.24–3.44, p = 0.006). Conclusion: Our results suggest that the CCND1 G870A single nucleotide polymorphism is associated with an increased risk of HCC in our Turkish population.

Cyclin D1b represses breast cancer cell growth by antagonizing the action of cyclin D1a on estrogen receptor α-mediated transcription

Alternate splicing of the cyclin D1 gene gives rise to transcripts a and b which encode two protein isoforms cyclin D1a and cyclin D1b. Cyclin D1a can substitute for estrogen to activate estrogen receptor alpha- (ERalpha) mediated transcription and can induce the proliferation of estrogen responsive tissues. However, little is known about the biological role of cyclin D1b in transcriptional regulation. In this study, we determined that cyclin D1b is incapable of inducing ERalpha-mediated transcription because it fails to recruit steroid receptor coactivator-1 (SRC-1) to ERalpha. Moreover, cyclin D1b antagonizes cyclin D1a-induced ERalpha-mediated transcription by competing with cyclin D1a for ERalpha binding. Cell proliferation assay showed that cyclin D1b repressed the ERalpha-positive breast cancer T47D cell growth. Our findings suggest that the cyclin D1b represses breast cancer cell growth by antagonizing the action of cyclin D1a on ERalpha-mediated transcription.