Full-length Cyclin Research Articles

Low-Molecular Weight Cyclin E Confers a Vulnerability to PKMYT1 Inhibition in Triple-Negative Breast Cancer.

Cyclin E is a regulatory subunit of CDK2 that mediates S phase entry and progression. The cleavage of full-length cyclin E (FL-cycE) to low-molecular weight isoforms (LMW-E) dramatically alters substrate specificity, promoting G1-S cell cycle transition and accelerating mitotic exit. Approximately 70% of triple-negative breast cancers (TNBC) express LMW-E, which correlates with poor prognosis. PKMYT1 also plays an important role in mitosis by inhibiting CDK1 to block premature mitotic entry, suggesting it could be a therapeutic target in TNBC expressing LMW-E. In this study, analysis of tumor samples of patients with TNBC revealed that coexpression of LMW-E and PKMYT1-catalyzed CDK1 phosphorylation predicted poor response to neoadjuvant chemotherapy. Compared with FL-cycE, LMW-E specifically upregulates PKMYT1 expression and protein stability, thereby increasing CDK1 phosphorylation. Inhibiting PKMYT1 with the selective inhibitor RP-6306 (lunresertib) elicited LMW-E-dependent antitumor effects, accelerating premature mitotic entry, inhibiting replication fork restart, and enhancing DNA damage, chromosomal breakage, apoptosis, and replication stress. Importantly, TNBC cell line xenografts expressing LMW-E showed greater sensitivity to RP-6306 than tumors with empty vector or FL-cycE. Furthermore, RP-6306 exerted tumor suppressive effects in LMW-E transgenic murine mammary tumors and patient-derived xenografts of LMW-E-high TNBC but not in the LMW-E null models examined in parallel. Lastly, transcriptomic and immune profiling demonstrated that RP-6306 treatment induced interferon responses and T-cell infiltration in the LMW-E-high tumor microenvironment, enhancing the antitumor immune response. These findings highlight the LMW-E/PKMYT1/CDK1 regulatory axis as a promising therapeutic target in TNBC, providing the rationale for further clinical development of PKMYT1 inhibitors in this aggressive breast cancer subtype. Significance: PKMYT1 upregulation and CDK1 phosphorylation in triple-negative breast cancer expressing low-molecular weight cyclin E leads to suboptimal responses to chemotherapy but sensitizes tumors to PKMYT1 inhibitors, proposing a personalized treatment strategy.

Abstract 308: Low molecular weight cyclin E deregulates DNA replication and damage repair to promote genomic instability in breast cancer

Abstract Background: Low molecular weight cyclin E (LMW-E) are oncogenic forms of cyclin E that are post translationally generated from the full-length cyclin E1 (FL-cycE). LMW-E is detected in breast cancer cells and tumor tissues, but not in normal mammary epithelial cells or adjacent normal tissues. Unlike FL-cycE, LMW-E drives mammary epithelial cell transformation in human cells and spontaneous mammary tumor formation in transgenic mouse models, but the oncogenic mechanisms of LMW-E and its unique function(s) independent of FL-cycE are not fully understood. It is currently assumed that LMW-E drives the tumorigenic process by promoting G1/S cell cycle transition and accelerating mitotic exit. Biochemical features such as longer protein half-life, higher affinity to its kinase partner CDK2, and resistance to endogenous CDK inhibitors such as p21 and p27 all promote the tumorigenic ability of LMW-E. Clinical studies in breast cancer reveal that overexpression of LMW-E predicts recurrence and poor survival in breast cancer patients independent of molecular subtype, Ki67 status, nodal status, or tumor grade, suggesting LMW-E may drive breast cancer development independent of its role in cell proliferation. In the current study, we tested the hypothesis that LMW-E promotes genomic instability by deregulating DNA replication and damage repair. Results: We generated immortalized pre-cancerous human mammary epithelial cells (hMECs) to express doxycycline inducible LMW-E or FL-cycE in CCNE1 knock-out background. We found that FL-cycE overexpression led to DNA replication stress and DNA damage accumulation, resulting in reduced cell viability. LMW-E overexpression, on the other hand, promoted cell survival under replication stress, resulting in persistent genomic instability. RNA-sequencing results showed LMW-E but not FL-cycE overexpression enhanced DNA replication and damage repair pathways. Molecularly, LMW-E interacted with and facilitated pre-replication complex assembly. LMW-E also mediated DNA repair by upregulating RAD51 and C17orf53, showing a dominant repairing effect over DNA damage induced by FL-cycE. Moreover, targeting the replication stress response pathway ATR-CHK1-RAD51 with small molecule inhibitors significantly decreased viability of LMW-E overexpressing hMECs and breast cancer cells. Lastly, we showed that positive LMW-E status was associated with genomic instability in tumors from a cohort of 725 patients diagnosed with early-stage breast cancer, further supporting our hypothesis that LMW-E promotes genomic instability to fuel breast cancer development. Conclusions: Collectively, our findings delineated a novel role for LMW-E in breast tumorigenesis mediated by replication stress tolerance and genomic instability, providing novel therapeutic strategies for LMW-E overexpressing breast cancers. Citation Format: Mi Li, Spiridon Tsavachidis, Fuchenchu Wang, Tuyen Bui, Tuyen D. Nguyen, Linjie Luo, Asha S. Multani, Melissa L. Bondy, Kelly K. Hunt, Khandan Keyomarsi. Low molecular weight cyclin E deregulates DNA replication and damage repair to promote genomic instability in breast cancer [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 308.

Low-molecular-weight cyclin E deregulates DNA replication and damage repair to promote genomic instability in breast cancer.

Low-molecular-weight cyclin E (LMW-E) is an N-terminus deleted (40 amino acid) form of cyclin E detected in breast cancer, but not in normal cells or tissues. LMW-E overexpression predicts poor survival in breast cancer patients independent of tumor proliferation rate, but the oncogenic mechanism of LMW-E and its unique function(s) independent of full-length cyclin E (FL-cycE) remain unclear. In the current study, we found LMW-E was associated with genomic instability in early-stage breast tumors (n = 725) and promoted genomic instability in human mammary epithelial cells (hMECs). Mechanistically, FL-cycE overexpression inhibited the proliferation of hMECs by replication stress and DNA damage accumulation, but LMW-E facilitated replication stress tolerance by upregulating DNA replication and damage repair. Specifically, LMW-E interacted with chromatin and upregulated the loading of minichromosome maintenance complex proteins (MCMs) in a CDC6 dependent manner and promoted DNA repair in a RAD51- and C17orf53-dependent manner. Targeting the ATR-CHK1-RAD51 pathway with ATR inhibitor (ceralasertib), CHK1 inhibitor (rabusertib), or RAD51 inhibitor (B02) significantly decreased the viability of LMW-E-overexpressing hMECs and breast cancer cells. Collectively, our findings delineate a novel role for LMW-E in tumorigenesis mediated by replication stress tolerance and genomic instability, providing novel therapeutic strategies for LMW-E-overexpressing breast cancers.

Abstract P2-05-02: Low molecular weight cyclin E facilitate replication stress tolerance in breast cancer development

Abstract Low Molecular Weight Cyclin E (LMW-E) are the tumor specific, oncogenic forms of cyclin E that are post translationally generated by neutrophil elastase (NE) mediated cleavage of the 50 KDa full-length cyclin E1 (FL-cycE, encoded by CCNE1 gene). While FL-cycE localizes mainly to the cell nucleus, LMW-E lack the N-terminus nuclear localization signal and are detected in both the nucleus and cytoplasm. Compared to FL-cycE, LMW-E exhibit longer half-life and higher affinity to their kinase partner CDK2 and are resistant to natural CDK inhibitors such as p21 and p27. It is currently assumed that LMW-E drive the tumorigenic process by promoting G1/S cell cycle transition and accelerating mitotic exit. Here we report that LMW-E overexpression also promotes genomic instability by deregulating DNA replication in a CDC6 dependent manner. To this end, we developed an immunohistochemistry (IHC) assay with a cyclin E antibody that can identify LMW-E expressing tumors and examined the association of genetic instability of each tumor with LMW-E status in 2 different cohorts of breast cancer patients. Cohort 1 is a retrospective cohort of 725 patients with stage I-II breast cancer treated at MD Anderson (Houston, TX) between 1985 and 1999. Cohort 2 is a prospective cohort of 85 patients with stage I-II breast cancer who enrolled in our study at MD Anderson between January 2000 and June 2010. Our results show that positive LMW-E status in stage 1 or 2 breast cancer patients correlates with increasing copy number variations, as identified by Molecular Inversion Probe (MIP) in cohort 1 and somatic mutations, as identified by Whole Exome Sequencing (WES) in cohort 2. Second, using immortalized human mammary epithelial cells (hMECs) engineered to express doxycycline inducible LMW-E or FL-cycE in CCNE1 knock-out background, we found that FL-cycE over-expression leads to DNA damage, cell cycle arrest and cell death. LMW-E overexpression, on the other hand, facilitates cell proliferation with damaged DNA, resulting in multi-nuclei and micro-nuclei formation in daughter cells. Third, overexpression of FL-cycE reduces chromatin bound MCM complex, while LMW-E overexpression promotes the chromatin loading of pre-replication complex including MCMs. Lastly, we show that LMW-E but not FL-cycE is the major form of cyclin E that binds to the chromatin. Specifically, in both LMW-Einducible hMECs and LMW-Ehighbreast tumor cell lines, CDC6 is required for the nuclear translocation and chromatin loading of LMW-E. Our findings have revealed the unique oncogenic function of LMW-E in deregulating replication licensing, promoting replication stress tolerance and genomic instability that fuels tumor development. These findings also provide potential novel therapeutic strategies for treating LMW-Ehigh breast tumors, who do not respond to the current standard of care therapies. Citation Format: Mi Li, Kwang Huei Low, Tuyen Bui, Kelly K Hunt, Khandan Keyomarsi. Low molecular weight cyclin E facilitate replication stress tolerance in breast cancer development [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-05-02.

Abstract P2-05-04: Low molecular weight cyclin E facilitates replication stress tolerance in breast cancer development

Abstract Purpose: Low Molecular Weight Cyclin E (LMW-E) are the oncogenic forms of cyclin E and were originally discovered in breast cancer (BC). LMW-E are generated from N-terminal cleavage of the 50 KDa, full-length cyclin E1 (FL-cycE). Our laboratory has established that LMW-E expression (i) correlates with poorer survival in BC patients; (ii) increases in frequency as BC progress from ductal carcinoma in situ to invasive ductal carcinoma; (iii) drives spontaneous and metastatic BC in murine transgenic models; (iii) causes human mammary epithelial cells (hMECs) to transform in vitro and form tumors in vivo. Elucidating the critical oncogenic functions and essential downstream factors of LMW-E is a current void in the field and may have important therapeutic implications. Experimental Design: Immunohistochemistry (IHC) analysis were performed using tumor specimens (n=725) from breast cancer patients diagnosed with Stage 1 or 2 disease to determine the level of LMW-E. Micro-dissected tumor DNA from these tissues were analyzed for copy number variations (CNVs) using Molecular Inversion Probe (MIP) based arrays. CCNE1(encoding Cyclin E1) knock-out 76NE6 and 76NF2V hMEC lines were generated by CRISPR, followed by transfection of lentivirus vector expressing doxycycline inducible EGFP fused LMW-E or FL-cycE. Time-lapse live cell imaging was performed to monitor the cell growth and phenotypes after induced expression of LWM-E or FL-cycE. DNA replication and replication stress were examined by BrdU labeling and immunofluorescent (IF) assays using antibodies against BrdU and Replication Protein A (RPA). IF for gamma-H2AX and western blotting for phosphor-RPA32, ATR-CHK1 and ATM-CHK2 pathways were performed to determine the DNA damage and responses. Results: Analysis of CNVs for the 725 tumors stratified by the cyclin E phenotypes reveal that the frequency of the CNVs (gains & losses) were the most significant in patients whose tumors expressed LMW-E and were predictive of poor prognosis independent of BC subtypes. DNA damage signals including gamma-H2AX foci and phosphor-CHK1 were similarly enhanced in hMECs induced for FL-cycE or LMW-E over-expression. Different effects of FL-cycE and LMW-E on DNA replication and replication stress were observed. Induced FL-cycE overexpression attenuated increased RPA foci and RPA phosphorylation, inhibits DNA replication and cell growth, and ultimately lead to cell death. LMW-E overexpression facilitated DNA replication and cell proliferation with damaged DNA, resulting in multi-nuclei and micro-nuclei formation in daughter cells. Conclusion: LMW-E expression positively correlates with genomic instability in BC samples. LMW-E facilitates hMECs to by-pass replication stress induced tumor barrier and survive with damaged DNA, resulting in abnormal nuclei formation, an oncogenic phenotype. Citation Format: Mi Li, Kwang Huei Low, Tuyen Bui, Kelly K Hunt, Khandan Keyomarsi. Low molecular weight cyclin E facilitates replication stress tolerance in breast cancer development [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-05-04.

Low-Molecular-Weight Cyclin E in Human Cancer: Cellular Consequences and Opportunities for Targeted Therapies.

Cyclin E, a regulatory subunit of cyclin-dependent kinase 2 (CDK2), is central to the initiation of DNA replication at the G1/S checkpoint. Tight temporal control of cyclin E is essential to the coordination of cell-cycle processes and the maintenance of genome integrity. Overexpression of cyclin E in human tumors was first observed in the 1990s and led to the identification of oncogenic roles for deregulated cyclin E in experimental models. A decade later, low-molecular-weight cyclin E (LMW-E) isoforms were observed in aggressive tumor subtypes. Compared with full-length cyclin E, LMW-E hyperactivates CDK2 through increased complex stability and resistance to the endogenous inhibitors p21CIP1 and p27KIP1 LMW-E is predominantly generated by neutrophil elastase-mediated proteolytic cleavage, which eliminates the N-terminal cyclin E nuclear localization signal and promotes cyclin E's accumulation in the cytoplasm. Compared with full-length cyclin E, the aberrant localization and unique stereochemistry of LMW-E dramatically alters the substrate specificity and selectivity of CDK2, increasing tumorigenicity in experimental models. Cytoplasmic LMW-E, which can be assessed by IHC, is prognostic of poor survival and predicts resistance to standard therapies in patients with cancer. These patients may benefit from therapeutic modalities targeting the altered biochemistry of LMW-E or its associated vulnerabilities. Cancer Res; 78(19); 5481-91. ©2018 AACR.

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.

Cyclin E Associates with the Lipogenic Enzyme ATP-Citrate Lyase to Enable Malignant Growth of Breast Cancer Cells.

Cyclin E is altered in nearly a third of invasive breast cancers where it is a powerful independent predictor of survival in women with stage I-III disease. Full-length cyclin E is posttranslationally cleaved into low molecular weight (LMW-E) isoforms, which are tumor-specific and accumulate in the cytoplasm because they lack a nuclear localization sequence. We hypothesized that aberrant localization of cytosolic LMW-E isoforms alters target binding and activation ultimately contributing to LMW-E-induced tumorigenicity. To address this hypothesis, we used a retrovirus-based protein complementation assay to find LMW-E binding proteins in breast cancer, identifying ATP-citrate lyase (ACLY), an enzyme in the de novo lipogenesis pathway, as a novel LMW-E-interacting protein in the cytoplasm. LMW-E upregulated ACLY enzymatic activity, subsequently increasing lipid droplet formation, thereby providing cells with essential building blocks to support growth. ACLY was also required for LMW-E-mediated transformation, migration, and invasion of breast cancer cells in vitro along with tumor growth in vivo In clinical specimens of breast cancer, the absence of LMW-E and low expression of adipophilin (PLIN2), a marker of lipid droplet formation, associated with favorable prognosis, whereas overexpression of both proteins correlated with a markedly worse prognosis. Taken together, our findings establish a novel relationship between LMW-E isoforms of cyclin E and aberrant lipid metabolism pathways in breast cancer tumorigenesis, warranting further investigation in additional malignancies exhibiting their expression. Cancer Res; 76(8); 2406-18. ©2016 AACR.

Abstract 3091: Targeting low molecular weight (LMW) cyclin E-Cdk2 pathway for the prevention of breast cancer

Abstract Our laboratory was first to discover that full length cyclin E is proteolytically cleaved into low-molecular weight isoforms (LMW-E) specifically in tumor cells. The LMW-E isoforms are hyperactive and over-expressed in 70% of all triple negative breast cancer (TNBC) compared to only 30% of non-TNBC patients. Overexpression of LMW-E in transgenic mice induces mammary tumors similar to the basal subtype of breast cancer, which accounts for 70% of all human TNBCs. We have reported that Cdk2 deficient mice were completely resistant to LMW-cyclin E mediated mammary tumors. In this study, we have interrogated the ability of roscovitine in preventing the development of triple negative mammary tumors in the MMTV-LMW-E; p53+/- mouse model. Initially, a group of mice were treated from age 3 months with vehicle (DMSO/PBS, 40:60v/v, N = 8) or roscovitine (100 mg/kg, N = 8) once every 4 and 5 days alternatively by intraperitoneal injection and were followed up for mammary tumor development. Mice did not show any signs of toxicity. In this schedule of treatment, we did not find any difference in time to tumor formation or survival between the 2 groups (8.3 vs 7.9 months, P = 0.70, and 8.9 vs 8.4 months, P = 0.98, respectively). However, we found a 2.8-fold increase in tumor doubling time in the roscovitine-treated group indicating that roscovitine treatment decreased tumor growth (12.1+/-4.4 vs 4.4+/-0.9 days, P<0.05). This was due to a decreased proliferation as measured by BrdU immunohistochemistry (IHC) (13.6+/-0.9% vs 30.0 +/- 7.7%, P<0.05) and an increased apoptotic index as measured by cleaved caspase 3 IHC (49.6+/-9.4 CC3+ cells/x40 field vs 25.9 +/- 9.9, P<0.05). A second group of mice were treated at age 3 months with the same schedule but were killed after 2 months of treatment and the mammary glands were collected to investigate the effects of drug treatment on normal and premalignant mammary cells. The BrdU staining showed that 2 months of roscovitine treatment caused a 3-fold decrease in the number of BrdU positive cells (5.4+/-1.5% vs 14.6+/-3.8%, P<0.05) but did not completely block cyclin E-induced proliferation. This result may explain why the roscovitine-treated mice developed tumor with similar kinetics as the control group. Our ongoing work is to use gene expression profiling to determine the RNA changes induced by roscovitine for the discovery of potential biomarkers. The results from the first trial is suggestive that the mice need to be treated on a continued schedule to ensure that proliferation of the cyclin E overexpressing mammary tissue is completely inhibited. To this end, we are now treating the mice twice a day, 7 days on, 21 days off. We predict that the new treatment schedule will be more efficacious in inhibiting tumor formation. These preliminary data could support the development of a clinical trial testing cyclin-dependent kinase inhibitors for the prevention of human triple negative breast cancer especially those with LMW-E overexpression. Citation Format: Said Akli, Dong Yang, Tuyen N. Bui, Khandan Keyomarsi. Targeting low molecular weight (LMW) cyclin E-Cdk2 pathway for the prevention of breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3091. doi:10.1158/1538-7445.AM2015-3091

Abstract P5-05-05: Low molecular weight cyclin E regulates response to aromatase inhibitors in post-menopausal breast cancer patients

Abstract Almost seventy percent of all breast cancer patients have estrogen receptor (ER) positive tumors requiring hormonal therapy. Aromatase inhibitors (AIs) are considered as the first line hormonal therapy for ER+ post-menopausal patients. However, resistance to these drugs remains a major challenge in clinic and the biology of such resistance is not clear. Previous studies have shown that cyclin E pathway, a key regulator in the G1 to S transition of cell cycle, is deregulated in breast cancer. Full-length cyclin E is abnormally cleaved into low molecular weight isoforms (LMW-E) that renders patients to poor survival. Here we hypothesize that cyclin E deregulation can confer resistance to AIs. To address this, we engineered aromatase overexpressing MCF7 cells to overexpress LMW-E under doxycycline inducible promoter. Full-length cyclin E, GFP and empty vector transfected cells were also generated and used as controls. Our results indicated that AIs inhibited proliferation by arresting the cells at G1 phase of the cell cycle. However, LMW-E expression significantly enhanced proliferation of the cells when treated with AIs. In addition, LMW-E bypassed G1 arrest following AI treatment. At the molecular level, AIs decreased CDK2, pCDK2, and Rb levels and attenuated Rb phosphorylation. However, these effects were completely rescued only when LMW-E was expressed. Moreover, using an in vitro kinase assay we indicated that AIs decreased CDK2 kinase activity while LMW-E expression reversed this effect by increasing CDK2 enzymatic activity. Taken together, these results suggest that LMW-E inactivates Rb protein as a tumor suppressor and renders the cells to bypass G1 checkpoint following AI treatment. In addition, this study provides early evidence that CDK2 inhibitors could be beneficial in combination with AIs for LMW-E expressing tumors. Currently we are investigating whether LMW-E can bypass the activity of AIs using inducible breast cancer cell line xenograft. We are also examining the correlation between cyclin E status and response to treatment in a cohort of patients who received AIs in the neo-adjuvant setting. Citation Format: Iman Doostan, Stacy L Moulder, Kelly K Hunt, Khandan Keyomarsi. Low molecular weight cyclin E regulates response to aromatase inhibitors in post-menopausal breast cancer patients [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-05-05.

Indole-3-carbinol and its N-alkoxy derivatives preferentially target ERα-positive breast cancer cells

Indole-3-carbinol (I3C) is a natural anti-carcinogenic compound found at high concentrations in Brassica vegetables. I3C was recently reported to inhibit neutrophil elastase (NE) activity, while consequently limiting the proteolytic processing of full length cyclin E into pro-tumorigenic low molecular weight cyclin E (LMW-E). In this study, we hypothesized that inhibition of NE activity and resultant LMW-E generation is critical to the anti-tumor effects of I3C. LMW-E was predominately expressed by ERα-negative breast cancer cell lines. However, ERα-positive cell lines demonstrated the greatest sensitivity to the anti-tumor effects of I3C and its more potent N-alkoxy derivatives. We found that I3C was incapable of inhibiting NE activity or the generation of LMW-E. Therefore, this pathway did not contribute to the anti-tumor activity of I3C. Gene expression analyzes identified ligand-activated aryl hydrocarbon receptor (AhR), which mediated sensitivity to the anti-tumor effects of I3C in ERα-positive MCF-7 cells. In this model system, the reactive oxygen species (ROS)-induced upregulation of ATF-3 and pro-apoptotic BH3-only proteins (e.g. NOXA) contributed to the sensitivity of ERα-positive breast cancer cells to the anti-tumor effects of I3C. Overexpression of ERα in MDA-MB-231 cells, which normally lack ERα expression, increased sensitivity to the anti-tumor effects of I3C, demonstrating a direct role for ERα in mediating the sensitivity of breast cancer cell lines to I3C. Our results suggest that ERα signaling amplified the pro-apoptotic effect of I3C-induced AhR signaling in luminal breast cancer cell lines, which was mediated in part through oxidative stress induced upregulation of ATF-3 and downstream BH3-only proteins.

Why are the truncated cyclin Es more effective CDK2 activators than the full-length isoforms?

Cell cycle regulating enzymes, CDKs, become activated upon association with their regulatory proteins, cyclins. The G1 cyclin, cyclin E, is overexpressed and present in low molecular weight (LMW) isoforms in breast cancer cells and tumor tissues. In vivo and in vitro studies have shown that these LMW isoforms of cyclin E hyperactivate CDK2 and accelerate the G1-S phase of cell division. The molecular basis of CDK2 hyperactivation due to LMW cyclin E isoforms in cancer cells is, however, unknown. Here, we employ a computational approach, combining homology modeling, bioinformatics analyses, molecular dynamics (MD) simulations, and principal component analyses to unravel the key structural features of CDK2-bound full-length and LMW isoforms of cyclin E1 and correlate those features to their differential activity. Results suggest that the missing N- and C-terminal regions of the cyclin E LMW isoforms constitute the Nuclear Localization Sequence (NLS) and PEST domains and are intrinsically disordered. These regions, when present in the full-length cyclin E/CDK2 complex, weaken the cyclin-CDK interface packing due to the loss of a large number of key interface interactions. Such weakening is manifested in the decreased contact area and increased solvent accessibility at the interface and also by the absence of concerted motions between the two partner proteins in the full-length complex. More effective packing and interactions between CDK2 and LMW cyclin E isoforms, however, produce more efficient protein-protein complexes that accelerate the cell division processes in cancer cells, where these cyclin E isoforms are overexpressed.

Hbo1 Is a Cyclin E/CDK2 Substrate That Enriches Breast Cancer Stem-like Cells

Expression of cyclin E proteolytic cleavage products, low-molecular weight cyclin E (LMW-E), is associated with poor clinical outcome in patients with breast cancer and it enhances tumorigenecity in mouse models. Here we report that LMW-E expression in human mammary epithelial cells induces an epithelial-to-mesenchymal transition phenotype, increases the CD44(hi)/CD24(lo) population, enhances mammosphere formation, and upregulates aldehyde dehydrogenase expression and activity. We also report that breast tumors expressing LMW-E have a higher proportion of CD44(hi)/CD24(lo) tumor cells as compared with tumors expressing only full-length cyclin E. In order to explore how LMW-E enriches cancer stem cells in breast tumors, we conducted a protein microarray analysis that identified the histone acetyltransferase (HAT) Hbo1 as a novel cyclin E/CDK2 substrate. The LMW-E/CDK2 complex phosphorylated Hbo1 at T88 without affecting its HAT activity. When coexpressed with LMW-E/CDK2, wild-type Hbo1 promoted enrichment of cancer stem-like cells (CSC), whereas the T88 Hbo1 mutant reversed the CSC phenotype. Finally, doxorubicin and salinomycin (a CSC-selective cytotoxic agent) synergized to kill cells expressing LMW-E, but not full-length cyclin E. Collectively, our results suggest that the heightened oncogenecity of LMW-E relates to its ability to promote CSC properties, supporting the design of therapeutic strategies to target this unique function.

Differential regulation of cyclin B1 degradation between the first and second meiotic divisions of bovine oocytes

During mammalian oocyte maturation, two consecutive meiotic divisions are required to form a haploid gamete. For each meiotic division, oocytes must transfer from metaphase to anaphase, but maturation promoting factor (cyclin-dependent kinase 1/cyclin B1) activity would keep the oocytes at metaphase. Therefore, inactivation of maturation promoting factor is needed to finish the transition and complete both these divisions; this is provided through anaphase-promoting complex/cyclosome-dependent degradation of cyclin B1. The objective of this study was to examine meiotic divisions in bovine oocytes after expression of a full length cyclin B1 and a nondegradable N-terminal 87 amino acid deletion, coupled with the fluorochrome Venus, by microinjecting their complementary RNA (cRNA). Overexpression of full-length cyclin B1-Venus inhibited homologue disjunction and first polar body formation in maturing oocytes (control 70% vs. overexpression 16%; P < 0.05). However at the same levels of expression, it did not block second meiotic metaphase and cleavage of eggs after parthenogenetic activation (control: 82% pronuclei and 79% cleaved; overexpression: 91% pronuclei and 89% cleaved). The full length cyclin B1 and a nondegradable N-terminal 87 amino acid deletion caused metaphase arrest in both meiotic divisions, whereas degradation of securin was unaffected. Roscovitine, a potent cyclin-dependent kinase 1 (CDK1) inhibitor, overcame this metaphase arrest in maturing oocytes at 140 μM, but higher doses (200 μM) were needed to overcome arrest in eggs. In conclusion, because metaphase I (MI) blocked by nondegradable cyclin B1 was distinct from metaphase II (MII) in their different sensitivities to trigger CDK1 inactivation, we concluded that mechanisms of MI arrest differed from MII arrest.

An Intron-Retaining Splice Variant of Human Cyclin A2, Expressed in Adult Differentiated Tissues, Induces a G1/S Cell Cycle Arrest In Vitro

BackgroundHuman cyclin A2 is a key regulator of S phase progression and entry into mitosis. Alternative splice variants of the G1 and mitotic cyclins have been shown to interfere with full-length cyclin functions to modulate cell cycle progression and are therefore likely to play a role in differentiation or oncogenesis. The alternative splicing of human cyclin A2 has not yet been studied.Methodology/Principal FindingsSequence-specific primers were designed to amplify various exon–intron regions of cyclin A2 mRNA in cell lines and human tissues. Intron retaining PCR products were cloned and sequenced and then overexpressed in HeLa cells. The subcellular localization of the splice variants was studied using confocal and time-lapse microscopy, and their impact on the cell cycle by flow cytometry, immunoblotting and histone H1 kinase activity. We found a splice variant of cyclin A2 mRNA called A2V6 that partly retains Intron 6. The gene expression pattern of A2V6 mRNA in human tissues was noticeably different from that of wild-type cyclin A2 (A2WT) mRNA. It was lower in proliferating fetal tissues and stronger in some differentiated adult tissues, especially, heart. In transfected HeLa cells, A2V6 localized exclusively in the cytoplasm whereas A2WT accumulated in the nucleus. We show that A2V6 induced a clear G1/S cell cycle arrest associated with a p21 and p27 upregulation and an inhibition of retinoblastoma protein phosphorylation. Like A2WT, A2V6 bound CDK2, but the A2V6/CDK2 complex did not phosphorylate histone H1.Conclusion/SignificanceThis study has revealed that some highly differentiated human tissues express an intron-retaining cyclin A2 mRNA that induced a G1/S block in vitro. Contrary to full-length cyclin A2, which regulates cell proliferation, the A2V6 splice variant might play a role in regulating nondividing cell states such as terminal differentiation or senescence.

Low molecular weight cyclin E is associated with p27-resistant, high-grade, high-stage and invasive bladder cancer

Expression of low molecular weight (LMW) isoforms of cyclin E is a strong predictor of poor outcome in patients with breast cancer. The purpose of this study was to examine the expression of full-length and LMW cyclin E in bladder cancer cell lines and patient tumors. We used western blotting, immunoprecipitation and kinase assays to examine the expression and activity of key cell cycle-regulatory proteins in various human bladder cell lines, both tumorigenic and non-tumorigenic. We also analyzed cyclin E expression, kinase activity and immune complex binding partners in 43 tissue samples from grade 2 and 3 transitional cell carcinomas. Cyclin E was overexpressed and LMW isoforms were present only in bladder cancer cells. Overexpression of LMW isoforms of cyclin E and increased cyclin E kinase activity were both significantly associated with tumorigenicity of the bladder cell lines (p = 0.005 and 0.022, respectively). Binding of the cyclin-dependent kinase inhibitors p21 and p27 to LMW cyclin E did not inhibit the kinase activity of cyclin E and cyclin-dependent kinase 2 in primary tumor samples overexpressing LMW cyclin E. Full-length and LMW cyclin E were significantly overexpressed in grade 3 tumors compared with grade 2 tumors (p = 0.004). Finally, LMW cyclin E levels were significantly associated with a non-papillary growth pattern (p = 0.031) and invasiveness (p = 0.021) of the bladder tumors and poor overall survival (p = 0.06). These results suggest that LMW cyclin E can be used as a new prognostic marker for bladder cancer.

Too Much Cleavage of Cyclin E Promotes Breast Tumorigenesis

Too Much Cleavage of Cyclin E Promotes Breast Tumorigenesis

LMW-E/CDK2 Deregulates Acinar Morphogenesis, Induces Tumorigenesis, and Associates with the Activated b-Raf-ERK1/2-mTOR Pathway in Breast Cancer Patients

Elastase-mediated cleavage of cyclin E generates low molecular weight cyclin E (LMW-E) isoforms exhibiting enhanced CDK2–associated kinase activity and resistance to inhibition by CDK inhibitors p21 and p27. Approximately 27% of breast cancers express high LMW-E protein levels, which significantly correlates with poor survival. The objective of this study was to identify the signaling pathway(s) deregulated by LMW-E expression in breast cancer patients and to identify pharmaceutical agents to effectively target this pathway. Ectopic LMW-E expression in nontumorigenic human mammary epithelial cells (hMECs) was sufficient to generate xenografts with greater tumorigenic potential than full-length cyclin E, and the tumorigenicity was augmented by in vivo passaging. However, cyclin E mutants unable to interact with CDK2 protected hMECs from tumor development. When hMECs were cultured on Matrigel, LMW-E mediated aberrant acinar morphogenesis, including enlargement of acinar structures and formation of multi-acinar complexes, as denoted by reduced BIM and elevated Ki67 expression. Similarly, inducible expression of LMW-E in transgenic mice generated hyper-proliferative terminal end buds resulting in enhanced mammary tumor development. Reverse-phase protein array assay of 276 breast tumor patient samples and cells cultured on monolayer and in three-dimensional Matrigel demonstrated that, in terms of protein expression profile, hMECs cultured in Matrigel more closely resembled patient tissues than did cells cultured on monolayer. Additionally, the b-Raf-ERK1/2-mTOR pathway was activated in LMW-E–expressing patient samples, and activation of this pathway was associated with poor disease-specific survival. Combination treatment using roscovitine (CDK inhibitor) plus either rapamycin (mTOR inhibitor) or sorafenib (a pan kinase inhibitor targeting b-Raf) effectively prevented aberrant acinar formation in LMW-E–expressing cells by inducing G1/S cell cycle arrest. LMW-E requires CDK2–associated kinase activity to induce mammary tumor formation by disrupting acinar development. The b-Raf-ERK1/2-mTOR signaling pathway is aberrantly activated in breast cancer and can be suppressed by combination treatment with roscovitine plus either rapamycin or sorafenib.

Abstract A50: Cyclin D1 isoforms selectively manipulate AR signaling to promote metastatic phenotypes in early and castration-resistant prostate cancer.

Abstract Cyclin D1 is a well-known inducer of cell cycle progression but has only recently been appreciated as a transcriptional regulator, controlling a milieu of transcriptional networks involved in both development and cancer. Intriguingly, while Cyclin D1 has only limited oncogenic activity, a variant that arises through oncogene induced alternative splicing (Cyclin D1b) has markedly enhanced oncogenic potential (as judged by in vitro and in vivo assays). Biochemical interrogation demonstrated that Cyclin D1b had little influence on cell cycle progression, indicating that oncogenic properties exerted by Cyclin D1b were likely transcriptionally regulated. Consonantly, it was shown that Cyclin D1b (but not full length Cyclin D1) is preferentially upregulated in malignant prostate cancers. Here, models were developed to determine the tumor-specific functions of Cyclin D1b, and unbiased analysis performed to assess cellular consequence. First, expression of Cyclin D1b was found to promote anchorage independent growth and enhance invasive potential in an androgen receptor (AR) dependent manner, while exerting no effect on cell cycle progression. Second, a large transcriptional network was determined to be uniquely under the control of Cyclin D1b, a subset of which are known AR target genes. Among this AR regulated gene set was the transcriptional regulator SNAI2 (SLUG), known to promote phenotypes associated with cancer progression and metastasis in various systems. Third, the molecular mechanisms underpinning Cyclin D1b function were revealed as Cyclin D1b enhanced AR occupancy at conserved AR bindings sites at SNAI2 regulatory loci, which correlated with chromatin modifications associated with active transcription. Fourth, molecular inhibition of SNAI2 levels was sufficient to inhibit D1b induced phenotypes both in vitro and in vivo. Finally, while both Cyclin D1b and SLUG are expressed in nearly 1/3 of primary prostate tumors, relative expression is increased in metastatic as well as castration resistant prostate cancer samples, indicating the Cyclin D1b/SLUG pathway is likely involved in disease progression. Together, the study to be presented defines a mechanism to explain Cyclin D1b-mediated effects in human disease, provides evidence of a direct AR driven gene that is induced exclusively during PCa progression, and identifies the Cyclin D1b/AR/SLUG axis as a major pathway associated with metastatic phenotypes. Citation Format: Michael A. Augello, Craig Burd, Ruth Bribe, Daniel Frigo, Jason Karch, Donald McDonnell, Tapio Visakorpi, Karen E. Knudsen. Cyclin D1 isoforms selectively manipulate AR signaling to promote metastatic phenotypes in early and castration-resistant prostate cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr A50.

P5-03-03: Identification of New Substrates for Breast Tumor Specific Low-Molecular-Weight Cyclin E Cyclin-Dependent-Kinase 2.

Abstract Background: Cyclin E overexpression occurs in 25% of breast cancer tumors and is linked to poor prognosis. In tumor cells full length cyclin E (FL-E) is processed by an elastase-like protease into low-molecular weight isoforms (LMW-E) that are biochemically hyperactive. When overexpressed in breast cancer cells, LMW-E induced genomic instability and resistance to p21, p27, and antiestrogens. LMW-E are tumor specific and are associated with the aggressive triple negative breast cancer (TNBC). Transgenic mice overexpressing LMW-E had increased incidence of mammary tumors and distant metastasis when compared to FL-E. Furthermore, when the role of CDK2 in LMW-E mediated mammary tumorigenesis was interrogated by crossing LMW-E transgenic mice with CDK2 knock out mice, we found that the mice are protected against mammary tumor formation. Additionally, treatment of LMW-E transgenic mice with roscovitine (a CDK2 inhibitor) delays mammary tumor formation. These results indicate that kinase inhibition may have therapeutic activity in LMW-E overexpressing tumors as shown in this preclinical model. These results lead us to hypothesize that the biological and biochemical differences between FL-E and LMW-E may be due to the phosphorylation of a distinct set of substrates when complexed with CDK2. Our goal is to identify distinct LMW-E/CDK2 substrates on a proteome-wide scale that could serve as novel therapeutic targets for the treatment of the aggressive LMW-E expressing TNBC. Material and Methods: We used two different approaches to identify LMW-E/CDK2 substrates. 1) We generated an analog sensitive CDK2 kinase, (F80A or F80G)-CDK2 to specifically radiolabel its substrates in cell extracts followed by their identification by mass spectroscopy. 2) We incubated ProtoArrayMicroarrays spotted with 3000 GST-tagged human protein on high density glass slides either with recombinant active FL-E/CDK2 or LMW-E/CDK2. Results: In the first approach, we expressed and purified wild-type CDK2 in complex with FL-E or LMW-E and CDK2 (F80A) and CDK2 (F80G) from insect cells. Although all 3 CDK2 kinases can use ATP to phosphorylate GST-Rb protein, only the F80G mutant can use PE-ATP-γ-S. In the second approach, we incubated protein arrays either with recombinant active FL-E/CDK2 or LMW-E/CDK2 at a concentration of 50 nM. Our first screen identified a total of 122 potential substrates to both FL-E/CDK2 or LMW-E/CDK2 kinase complexes. We only identified 1 protein that is phosphorylated by FL-E/CDK2 significantly more than by LMW-E/CDK2 as compared to the 32 potential substrates specific to LMW-E/CDK2 suggesting that by losing the N-terminal portion, the LMW-E/CDK2 kinase complex is able to specifically interact and phosphorylate novel proteins. Discussion: The identification of new physiological LMW-E/CDK2 substrates will lead to the development of novel targets for therapeutics and the identification of the biological function for the treatment of the aggressive LMW-E expressing TNBC. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-03-03.