Cell Cycle Components Research Articles

Antiproliferative activity of goniothalamin enantiomers involves DNA damage, cell cycle arrest and apoptosis induction in MCF-7 and HB4a cells

(R)-goniothalamin (R-GNT) is a styryl lactone that exhibits antiproliferative property against several tumor cell lines. (S)-goniothalamin (S-GNT) is the synthetic enantiomer of R-GNT, and their biological properties are poorly understood. The aim of this study was to evaluate the antiproliferative mechanisms of (R)-goniothalamin and (S)-goniothalamin in MCF-7 breast cancer cells and HB4a epithelial mammary cells. To determine the mechanisms of cell growth inhibition, we analyzed the ability of R-GNT and S-GNT to induce DNA damage, cell cycle arrest and apoptosis. Moreover, the gene expression of cell cycle components, including cyclin, CDKs and CKIs, as well as of genes involved in apoptosis and the DNA damage response were evaluated. The natural enantiomer R-GNT proved more effective in both cell lines than did the synthetic enantiomer S-GNT, inhibiting cell proliferation via cell cycle arrest and apoptosis induction, likely in response to DNA damage. The cell cycle inhibition caused by R-GNT was mediated through the upregulation of CIP/KIP cyclin-kinase inhibitors and through the downregulation of cyclins and CDKs. S-GNT, in turn, was able to cause G0/G1 cell cycle arrest and DNA damage in MCF-7 cells and apoptosis induction only in HB4a cells. Therefore, goniothalamin presents potent antiproliferative activity to breast cancer cells MCF-7. However, exposure to goniothalamin brings some undesirable effects to non-tumor cells HB4a, including genotoxicity and apoptosis induction.

Cell type-specific translational repression of Cyclin B during meiosis in males

The unique cell cycle dynamics of meiosis are controlled by layers of regulation imposed on core mitotic cell cycle machinery components by the program of germ cell development. Although the mechanisms that regulate Cdk1/Cyclin B activity in meiosis in oocytes have been well studied, little is known about the trans-acting factors responsible for developmental control of these factors in male gametogenesis. During meiotic prophase in Drosophila males, transcript for the core cell cycle protein Cyclin B1 (CycB) is expressed in spermatocytes, but the protein does not accumulate in spermatocytes until just before the meiotic divisions. Here, we show that two interacting proteins, Rbp4 and Fest, expressed at the onset of spermatocyte differentiation under control of the developmental program of male gametogenesis, function to direct cell type- and stage-specific repression of translation of the core G2/M cell cycle component cycB during the specialized cell cycle of male meiosis. Binding of Fest to Rbp4 requires a 31-amino acid region within Rbp4. Rbp4 and Fest are required for translational repression of cycB in immature spermatocytes, with Rbp4 binding sequences in a cell type-specific shortened form of the cycB 3' UTR. Finally, we show that Fest is required for proper execution of meiosis I.

Cell type-specific translational repression of Cyclin B during meiosis in males.

The unique cell cycle dynamics of meiosis are controlled by layers of regulation imposed on core mitotic cell cycle machinery components by the program of germ cell development. Although the mechanisms that regulate Cdk1/Cyclin B activity in meiosis in oocytes have been well studied, little is known about the trans-acting factors responsible for developmental control of these factors in male gametogenesis. During meiotic prophase in Drosophila males, transcript for the core cell cycle protein Cyclin B1 (CycB) is expressed in spermatocytes, but the protein does not accumulate in spermatocytes until just before the meiotic divisions. Here, we show that two interacting proteins, Rbp4 and Fest, expressed at the onset of spermatocyte differentiation under control of the developmental program of male gametogenesis, function to direct cell type- and stage-specific repression of translation of the core G2/M cell cycle component cycB during the specialized cell cycle of male meiosis. Binding of Fest to Rbp4 requires a 31-amino acid region within Rbp4. Rbp4 and Fest are required for translational repression of cycB in immature spermatocytes, with Rbp4 binding sequences in a cell type-specific shortened form of the cycB 3' UTR. Finally, we show that Fest is required for proper execution of meiosis I.

Dickkopf-3 (DKK-3) obstructs VEGFR-2/Akt/mTOR signaling cascade by interacting of β2-microglobulin (β2M) in ovarian tumorigenesis

In this study, we investigated a possible mechanism of β2-microglobulin (β2M) function in cancer metastases in vitro, using a human ovarian carcinoma cell line. β2M, a modulator acts as a cell growth-promoting and cellular signaling factors, was identified as a dickkopf-3 (DKK-3) interacting protein. We also observed that DKK-3 suppresses endothelial cell angiogenesis of β2M through vascular endothelial growth factor receptor-2 (VEGFR-2) in tumorigenesis. Luciferase activity was remarkably reduced by the transfection of DKK-3 in a dose-dependent manner. In addition, over-expression of β2M activates cell growth by suppressing DKK-3-induced apoptosis. The effect of β2M on cell cycle and apoptosis-regulatory components was also confirmed through the silencing of β2M expression. Furthermore, induction of β2M-mediated VEGFR-2/Akt/mTOR phosphorylation and tumor angiogenesis was significantly suppressed by over-expression of DKK-3. Taken together, our results suggest an underlying mechanism for an increase of β2M-related activity in ovarian tumor cells.

Abstract 1844: Genome wide analysis of AR-cell cycle interplay reveals novel functions in cancer

Abstract The androgen receptor (AR) plays a vital role in prostate cancer (PCa) due in part to its ability to interact with cell cycle components in order to drive cell cycle transition. Numerous points of cross-talk have been identified, wherein specific components of the cell cycle machinery “feed back” to modulate AR function, and these interactions are thought to be altered in human malignancies. Despite these observations, the majority of genome wide analyses for AR have been performed in cells that have exited cell cycle (G0). Here, the cell cycle dependent AR transcriptome and cistrome was identified, revealing new and unexpected functions for AR in cycling tumor cells. In studies to be discussed, cells were arrested in 5 distinct phases of the cell cycle, stimulated with androgen, and AR activity assessed through gene expression and ChIP-Seq analyses. In AR binding analyses, significant overlap was seen with previously identified sites, but were accompanied by novel binding events that could be segregated into those that are specific to cycling cells and occur in all phases (“cell cycle common”) or show cell cycle stage specific binding (“phase exclusive”). Over 50% of the cell cycle common sites, and up to 95% of the phases exclusive sites were novel AR occupied sites. Additionally, using a “guilty by association” approach to determine potentially AR regulated genes from this novel cistromic data, it was determined that close to 50% of cell cycle common, and 70% of phase exclusive binding uncover novel candidates for AR regulation. Cistrome data was therefore overlaid with microarray data, to prioritize discovery of meaningful, cell cycle specific AR binding events. Analyses to be discussed reveal striking new insight into disease relevant AR function. In sum, these data rigorously demonstrate that AR acts in a cell cycle dependent manner, and that these functions of AR have a major impact on tumor cell phenotypes. Citation Format: Christopher McNair, Jonathan Goodwin, Michael Augello, Alfonso Urbanucci, Matthew Schiewer, Clay Comstock, Adam Ertel, Liguo Wang, Qianben Wang, Ian Mills, Wei Li, Jason Carroll, Karen Knudsen. Genome wide analysis of AR-cell cycle interplay reveals novel functions in 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 1844. doi:10.1158/1538-7445.AM2015-1844

Abstract A44: Targeting cancer-associated fibroblasts in pancreatic ductal adenocarcinoma: Identification and characterization of therapeutic candidates

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease with an overall survival rate of less than 5%. One of the main reasons for the poor prognosis of PDAC is thought to be due to its abundant fibrotic stromal tissue, which may act as a barrier for drug delivery, thus resulting in inefficient therapeutic treatments. Moreover, it is believed that the interactions between tumor and stromal cells play a very important role in disease progression. In this complex tumor microenvironment, cancer associated fibroblasts (CAFs) are the most significant cell type responsible for the production of extracellular matrix proteins resulting in increased stiffness of the tumor. Therefore, targeting CAFs in PDAC represents a promising therapeutic approach. In order to find attractive candidates to target the stromal tissue, we have isolated CAFs from our K-Ras driven PDAC mouse model [K-Ras+/LSLG12V;p53lox/lox;Elas-tTA/TetO-Cre strain] by cell sorting taking advantage of the high levels of expression of the PGDF Receptor-Alpha in this cell type. Next, we performed gene expression profiling by RNA sequencing comparing isolated normal pancreatic fibroblasts and CAFs. In order to complete our findings and direct our attention to tumor stromal communication we have also sequenced the transcriptome of the correspondent tumoral cells. Differential expression analysis showed significant upregulation in CAFs of a variety of functionally cancer associated cellular processes such as extracellular matrix remodeling, growth factor induced tumor growth, invasion and metastatic dissemination. Moreover, we observed a strong pro-inflammatory gene signature as well as upregulated chemokine, cytokine and adipocytokine signaling and amyloidosis in CAFs compared to normal fibroblasts through complement and coagulation cascade activation. Regulation of the inflammatory response included hyperactivity of AP1/JNK, IL6, Toll-like Receptor and NFKB pathways. However, we detected downregulation of cell cycle components, metabolic pathways and a significant decrease in the expression of p53 pathway related genes, an observation that has been already reported in CAFs associated with other cancer types. Connecting pathway mapping by Gene Set Enrichment Analysis to a novel method for generation and integration of druggable genes by cell-drug interactions offered a pool of candidates for final selection of our therapeutic approach. To further investigate the molecular mechanisms that control the fibroinflammatory stroma in PDAC, we have validated this signature by qRT-PCR in a set of CAFs and tumor samples derived from our tumor model as well as from human patients. Functional validation in vitro and in vivo of candidate genes derived from this experimental approach should lead us to identify new therapeutic targets against this deadly disease by targeting tumor-stromal cell interactions. Citation Format: Magdolna Djurec, Osvaldo Grana, David Pisano, Albert Lee, Raul Rabadan, Carmen Guerra, Mariano Barbacid. Targeting cancer-associated fibroblasts in pancreatic ductal adenocarcinoma: Identification and characterization of therapeutic candidates. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A44.

Dynein Activity Induced by a Kunitz‐type Molecule Acting on the Proteasome

MethodsCell culture: Mia PaCa‐2 and SK‐Mel‐28 tumor cells. Western blotting: Proteins were analyzed via chemiluminescence. Confocal microscopy: Live or fixed cells were analyzed using labeled Amblyomin‐X or specific antibodies. Proteasome assay: Trypsin and chymotrypsin‐like activities were accessed using chromogenic substrates. Co‐IP: Cell lysates were submitted to Co‐IP and injected into a mass spectrometer.ResultsProteasome inhibition by Amblyomin‐X and its cellular uptake was abolished when the cells were pretreated with a dynein inhibitor. Amblyomin‐X increased Rab11A protein levels and induced its co‐localization with dynein. Several proteins were found bound to dynein, such as receptors, signaling transducers, early and recycling endosome components, proteasome, aggresome, ubiquitin, cell cycle, apoptosis and extracellular matrix components.ConclusionsDynein was required for Amblyomin‐X uptake and may transport the endosome towards the endocytic recycling compartment, because Rab11A was overexpressed, co‐localized with dynein; and Rab11A and Rab20 but not late endosome markers were bound to dynein. In addition, dynein was required for proteasome inhibition induced by Amblyomin‐X, thus suggesting its transportation to the molecular target. Some proteins, such as Bag3 and Raptor were found bound to dynein and this data corroborates with the aggresome formation and autophagy inhibition induced by Amblyomin‐X, as previously reported. Furthermore, dynein ligands indicated a possible involvement of the lipid rafts, intracellular pathways, transcription factors and membrane receptors. Those findings distinguish this molecule from proteasome inhibitors, such as bortezomib and carlfizomib. Supported by FAPESP.

Kif18a is specifically required for mitotic progression during germ line development

Genome integrity in the developing germ line is strictly required for fecundity. In proliferating somatic cells and in germ cells, there are mitotic checkpoint mechanisms that ensure accurate chromosome segregation and euploidy. There is growing evidence of mitotic cell cycle components that are uniquely required in the germ line to ensure genome integrity. We previously showed that the primary phenotype of germ cell deficient 2 (gcd2) mutant mice is infertility due to germ cell depletion during embryogenesis. Here we show that the underlying mutation is a mis-sense mutation, R308K, in the motor domain of the kinesin-8 family member, KIF18A, a protein that is expressed in a variety of proliferative tissues and is a key regulator of chromosome alignment during mitosis. Despite the conservative nature of the mutation, we show that its functional consequences are equivalent to KIF18A deficiency in HeLa cells. We also show that somatic cells progress through mitosis, despite having chromosome alignment defects, while germ cells with similar chromosome alignment defects undergo mitotic arrest and apoptosis. Our data provide evidence for differential requirements for chromosome alignment in germ and somatic cells and show that Kif18a is one of a growing number of genes that are specifically required for cell cycle progression in proliferating germ cells.

Teriflunomide, an immunomodulatory drug, exerts anticancer activity in triple negative breast cancer cells.

Triple-negative breast cancer (TNBC) is defined as a group of primary breast cancers lacking expression of estrogen, progesterone, and human epidermal growth factor receptor-2 (HER-2) receptors, characterized by higher relapse rate and lower survival compared with other subtypes. Due to lack of identified targets and molecular heterogeneity, conventional chemotherapy is the only available option for treatment of TNBC, but non-discordant positive therapeutic efficacy could not be achieved. Here, we demonstrated that these TNBC cells were sensitive to teriflunomide, which was a well-known immunomodulatory drug for treatment of relapsing multiple sclerosis (MS). Potent anti-cancer effects in TNBC invitro, including proliferation inhibition, cell cycle delay, cell apoptosis, and suppression of cell motility and invasiveness, could be achieved with this agent. Of note, we showed that multiple signals involved in TNBC proliferation, survival, migratory, and invasive potential were under regulation by teriflunomide. Among them, we identified down-regulation of growth factor receptors to abolish growth maintenance, suppression of c-Myc, and cyclin D1 to contribute to its anti-proliferative effect, modulation of components of cell cycle to induce S-phase arrest, degradation of Bcl-xL, and up-regulation of BAX via activation of MAPK pathway to induce apoptosis, and inhibition of epithelial-mesenchymal transition (EMT) process, matrix metalloproteinase-9 (MMP9) expression, and inactivation of Src/FAK to reduce TNBC migration and invasion. The results identified teriflunomide may be of therapeutic benefit for the more aggressive and difficult-to-treat breast cancer subtype, indicating the use of teriflunomide for clinical trials for treatment of TNBC patients.

Lung Cancer Transcriptomes Refined with Laser Capture Microdissection

We evaluated the importance of tumor cell selection for generating gene signatures in non-small cell lung cancer. Tumor and nontumor tissue from macroscopically dissected (Macro) surgical specimens (31 pairs from 32 subjects) was homogenized, extracted, amplified, and hybridized to microarrays. Adjacent scout sections were histologically mapped; sets of approximately 1000 tumor cells and nontumor cells (alveolar or bronchial) were procured by laser capture microdissection (LCM). Within histological strata, LCM and Macro specimens exhibited approximately 67% to 80% nonoverlap in differentially expressed (DE) genes. In a representative subset, LCM uniquely identified 300 DE genes in tumor versus nontumor specimens, largely attributable to cell selection; 382 DE genes were common to Macro, Macro with preamplification, and LCM platforms. RT-qPCR validation in a 33-gene subset was confirmatory (ρ=0.789 to 0.964, P = 0.0013 to 0.0028). Pathway analysis of LCM data suggested alterations in known cancer pathways (cell growth, death, movement, cycle, and signaling components), among others (eg, immune, inflammatory). A unique nine-gene LCM signature had higher tumor-nontumor discriminatory accuracy (100%) than the corresponding Macro signature (87%). Comparison with Cancer Genome Atlas data sets (based on homogenized Macro tissue) revealed both substantial overlap and important differences from LCM specimen results. Thus, cell selection via LCM enhances expression profiling precision, and confirms both known and under-appreciated lung cancer genes and pathways.

Lysyl oxidase-like 2 (LOXL2) controls tumor-associated cell proliferation through the interaction with MARCKSL1

Lysyl oxidase-like 2 (LOXL2) is a member of the lysyl oxidase gene family that contributes to the invasiveness and metastasis in tumor progression. However, the role of LOXL2 in cellular signaling is incompletely understood. In this study, we investigated a possible mechanism of LOXL2 function in tumor metastases in vitro, using a human breast carcinoma cell line. Myristoylated alanine-rich C kinase substrate-like 1 (MARCKSL1), a modulator in the regulation of cellular homeostasis, was identified as a LOXL2 interacting protein. We examined the binding domains that are required for the interaction between LOXL2 and MARCKSL1. The scavenger-receptor domain of LOXL2 was shown to interact with the N-terminal domain of MARCKSL1. Luciferase activity was noticeably reduced by the transfection of MARCKSL1 in a dose-dependent manner. In addition, over-expression of LOXL2 activates cell growth by inhibiting MARCKSL1-induced apoptosis. The effect of LOXL2 on cell cycle and apoptosis-related components was also confirmed through the silencing of LOXL2 expression. LOXL2 activates the FAK/Akt/mTOR signaling pathways, and MARCKSL1 suppresses LOXL2-induced oncogenesis. These insights supply evidence that LOXL2 promotes cell proliferation and inhibits apoptotic cell death. Taken together, our results indicate an underlying mechanism for an increase of LOXL2-related activity in breast tumor cells.

MicroRNAs 424 and 503 are mediators of the anti-proliferative and anti-invasive action of the thyroid hormone receptor beta.

The thyroid hormone receptors (TRs) mediate tumor suppressive effects in hepatocarcinoma and breast cancer cells. Here we show that incubation of hepatocarcinoma SK-hep1 cells expressing TRb with the thyroid hormone T3 induces transcription of the polycistronic message coding for microRNAs 424 and 503. TRb binds to the promoter region of these miRNAs and T3 induces an exchange of corepressors and coactivators inducing histone acetylation and transcriptional stimulation. We have validated cell cycle components as targets of these miRNAs. Overexpression of miR-424 mimicked the repressive effect of T3 on cell proliferation, growth in suspension, migration and invasion. Knockdown of miR-424 or miR-503 reduced the inhibitory effect of the hormone. T3 increased miR-424 and miR-503 in breast cancer cells expressing TRb, and this induction is also involved in the anti-invasive effects of the hormone. Furthermore, miR-424 or miR-503 depletion enhanced extravasation to the lungs of hepatocarcinoma cells injected in the tail vein of mice. The levels of these miRNAs were reduced in xenograft tumors formed in hypothyroid nude mice that are more invasive. Therefore, miR-424 or miR-503 mediate anti-proliferative and anti-invasive actions of TRb both in cultured cells and in vivo.

Oncogenic potential of CK2α and its regulatory role in EGF‐induced HDAC2 expression in human liver cancer

Histone deacetylase 2 (HDAC2) is aberrantly regulated and plays a pivotal role in the development of hepatocellular carcinoma (HCC) through regulation of cell-cycle components at the transcriptional level, but the underlying mechanism leading to oncogenic HDAC2 remains unknown. In this study, we show that expression of CK2α (casein kinase II α subunit) was up-regulated in a large cohort of human HCC patients, and that high expression of CK2α was significantly associated with poor prognosis of HCC patients in terms of five-year overall survival. It was also found that CK2α over-expression positively correlated with HDAC2 over-expression in a subset of HCCs. We observed that treatment with epidermal growth factor (EGF) elicited an increase in CK2α expression and Akt phosphorylation, causing induction of HDAC2 expression in liver cancer cells. It was also observed that ectopic expression of dominant-negative CK2α blocked EGF-induced HDAC2 expression, and that ectopic CK2α expression attenuated the suppressive effect of Akt knockdown on HDAC2 expression in liver cancer cells. Targeted disruption of CK2α influenced the cell cycle, causing a significant increase in the number of liver cancer cells remaining in G₂/M phase, and suppressed growth via repression of Cdc25c and cyclin B in liver cancer cells. Taken together, our findings suggest the oncogenic potential of CK2α in liver tumorigenesis. Furthermore, a regulatory mechanism for HDAC2 expression is proposed whereby EGF induces transcriptional activation of HDAC2 by CK2α/Akt activation in liver cancer cells. Therefore, this makes CK2α a promising target in cancer therapy.

Drosophila Embryonic Cell-Cycle Mutants

Nearly all cell division mutants in Drosophila were recovered in late larval/pupal lethal screens, with less than 10 embryonic lethal mutants identified, because larval development occurs without a requirement for cell division. Only cells in the nervous system and the imaginal cells that generate the adult body divide during larval stages, with larval tissues growing by increasing ploidy rather than cell number. Thus, most mutants perturbing mitosis or the cell cycle do not manifest a phenotype until the adult body differentiates in late larval and pupal stages. To identify cell-cycle components whose maternal pools are depleted in embryogenesis or that have specific functions in embryogenesis, we screened for mutants defective in cell division during embryogenesis. Five new alleles of Cyclin E were recovered, ranging from a missense mutation that is viable to stop codons causing embryonic lethality. These permitted us to investigate the requirements for Cyclin E function in neuroblast cell fate determination, a role previously shown for a null Cyclin E allele. The mutations causing truncation of the protein affect cell fate of the NB6-4 neuroblast, whereas the weak missense mutation has no effect. We identified mutations in the pavarotti (pav) and tumbleweed (tum) genes needed for cytokinesis by a phenotype of large and multinucleate cells in the embryonic epidermis and nervous system. Other mutations affecting the centromere protein CAL1 and the kinetochore protein Spc105R caused mitotic defects in the nervous system.

Cell Cycle Regulation During Neurogenesis in the Embryonic and Adult Brain

Throughout the development of the central nervous system, neural crest cells and the primary neural stem cells originate several non-neuronal and neuronal cell types. Undifferentiated stem cells exist in the adult brain, mainly in the dentate gyrus of the hippocampus and in the subventricular zone of the lateral ventricles, and can produce new neurons, participating in brain plasticity and tissue regeneration. Neurogenesis in the embryonic and adult brain occurs under the control of intrinsic and extrinsic factors. However, the mechanisms, by which cell cycle components control neural stem cell proliferation and consequently neurogenesis, still lack further investigation. We discuss here recent knowledge obtained on cell cycle components as key regulators of neural stem and progenitor cell proliferation and differentiation in the embryonic and adult brain.

Disparities in breast cancer outcomes between Caucasian and African American women: a model for describing the relationship of biological and nonbiological factors.

Breast cancer is the most common malignancy in women in the United States but significant disparities exist for African American women compared to Caucasian women. African American women present with breast cancer at a younger age and with a greater incidence under the age of 50 years, develop histologically more aggressive tumors that are at a more advanced stage at presentation, and have a worse disease-free and overall survival than Caucasian women. The biological characteristics of the primary tumor play an important role in determining the outcome of the disparity, and significant differences have been identified between African American and Caucasian breast cancer in steroid receptor and growth factor receptor content, mutations in cell cycle components, chromosomal abnormalities, and tumor suppressor and other cancer genes. The consequences of the biological factors are influenced by a variety of nonbiological factors, including socioeconomic, health care access, reproductive, and confounding factors. The nonbiological factors may act directly to enhance (or inhibit) the consequences of the biological changes, indirectly to facilitate outcome of the disparity, or as a cofounding factor, driving the association between the biological factors and the disparity. The prevention and management of the disparities will require an understanding of the relationship of biological and nonbiological factors. The present review was undertaken to promote this understanding by describing the biological basis of the four major disparities - early age of onset, more advanced stage of disease, more aggressive histologic changes, and worse survival - and the important relationship to the nonbiological factors. A model is proposed to provide a comprehensive view of this relationship, with the goal of facilitating an understanding of each disparity and the issues that need to be addressed to eliminate the disparity.

Abstract 3574: Studies of castrate resistant 22Rv1 cells identifies AR regulated interrelated networks of transcription factors, co-regulators, chromatin, and nuclear scaffolding proteins.

Abstract Prostate cancer development, progression and acquisition of castrate resistance are reliant on the activity of the androgen receptor (AR), a transcription factor that governs the proliferation of prostate cancer cells. Multiple studies have focused on the principle components that are critical for cell cycle transversal and these studies have identified members of the E2F/RB family, c-Myc, cyclins and cyclin dependent kinase inhibitors as key proteins that are instrumental in prostate cell proliferation and development of castrate resistance. But the mechanism by which the AR dominates this process is unclear. To identify the link between the AR and cell cycle control we used 22Rv1 cells which are castrate resistant due to the expression of low molecular weight AR forms that are missing the ligand binding domain. However, a siRNA mediated decrease in AR expression induces a growth arrest confirming that proliferation is AR dependent. RNA-seq analysis of gene expression at castrate levels of androgen and following AR ablation identified transcripts that were AR dependent. This methodology reduced the number of AR regulated transcripts, by excluding transcripts regulated by super-physiological levels of androgens. The AR regulated transcripts included previously identified genes (PMEPA1, C1orf116, PCDH7, APP) including NFAT a gene shown to be regulated by a low molecular weight AR isoform. The analysis also revealed that interrelated networks of transcription factors and co-factors were AR transactivated or repressed. These included proteins such as the LBR, LMNB, TMPO and SATB1 that do not directly promote transcription, but rather link chromatin to the nuclear membrane or scaffolding, suggesting that the AR may modulate gene expression through epigenetics mechanisms. Network analysis showed connections between these proteins and cell cycle components including E2Fs and Myc where a number of the regulated transcripts are known E2F or Myc targets. In addition, this analysis identified a number of non-coding RNAs; some that have been previously described and some that have yet to be annotated. qPCR studies found that a number of the transcripts regulated in 22Rv1 cells are also AR regulated in LNCaP cells. Coupled with ChIP-seq data, the study characterizes the link between the AR and the cell cycle machinery and chromatin modulating proteins. Citation Format: Maria Mudryj, Stephen J. Libertini, Alan P. Lombard. Studies of castrate resistant 22Rv1 cells identifies AR regulated interrelated networks of transcription factors, co-regulators, chromatin, and nuclear scaffolding proteins. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3574. doi:10.1158/1538-7445.AM2013-3574

Abstract 5173: The circadian factor period 2 modulates p53 stability and function in downstream signaling.

Abstract The human Period 2 (hPer2) factor is a transcriptional regulator placed at the core of the circadian clock mechanism responsible for generating the negative feedback loop that sustains the clock. Its relevance to human diseases is underlined by alterations in its function that impacts many biochemical and physiological processes and, when absent, results in the development of various cancers. The tumor suppressor role of Per2 is speculated to involve transcriptional activation of p53, altered expression of cell cycle components, and regulation of the DNA-damage response pathway. However, it is entirely unclear how Per2 operates mechanistically. First, we identified hPer2 binds the C-terminus half of human p53 (hp53) and forms a stable trimeric complex with hp53’s negative regulator, the oncogenic protein Mdm2. Second, we determined that hPer2 binding to hp53 prevents Mdm2 from ubiquitinating and targeting hp53 by the proteasome. Accordingly, downregulation of hPer2 expression directly impacts hp53 levels whereas its overexpression influences both hp53 protein stability and transcription. Furthermore, we spatially define the distribution of the trimeric complex and determine the site for processing to be located in the nucleus. Third, we establish that hp53-mediated gene transcription is influenced by the presence of hPer2. Target genes such as 14-3-3σ, hp21WAF1/CIP1, and gadd45α show a synergistic increase in expression when hPer2 and hp53 are co-expressed in a hp53-deficient background. This result is the direct consequence of hPer2 dissociation of hp53 as result of checkpoint activation as shown by studies performed using a constitutively bound form of the hPer2/hp53 complex. Overall, our findings directly place hPer2 at the heart of the hp53-mediated response by modulating its stability and controlling its function. Citation Format: Carla V. Finkielstein, Tetsuya Gotoh, Marian Vila-Caballer, carlo santos, jingjing liu, jianhua yang. The circadian factor period 2 modulates p53 stability and function in downstream signaling. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5173. doi:10.1158/1538-7445.AM2013-5173 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Scratch2 Prevents Cell Cycle Re-Entry by Repressing miR-25 in Postmitotic Primary Neurons

During the development of the nervous system the regulation of cell cycle, differentiation, and survival is tightly interlinked. Newly generated neurons must keep cell cycle components under strict control, as cell cycle re-entry leads to neuronal degeneration and death. However, despite their relevance, the mechanisms controlling this process remain largely unexplored. Here we show that Scratch2 is involved in the control of the cell cycle in neurons in the developing spinal cord of the zebrafish embryo. scratch2 knockdown induces postmitotic neurons to re-enter mitosis. Scratch2 prevents cell cycle re-entry by maintaining high levels of the cycle inhibitor p57 through the downregulation of miR-25. Thus, Scratch2 appears to safeguard the homeostasis of postmitotic primary neurons by preventing cell cycle re-entry.

Re-cycling Paradigms: Cell Cycle Regulation in Adult Hippocampal Neurogenesis and Implications for Depression

Since adult neurogenesis became a widely accepted phenomenon, much effort has been put in trying to understand the mechanisms involved in its regulation. In addition, the pathophysiology of several neuropsychiatric disorders, such as depression, has been associated with imbalances in adult hippocampal neurogenesis. These imbalances may ultimately reflect alterations at the cell cycle level, as a common mechanism through which intrinsic and extrinsic stimuli interact with the neurogenic niche properties. Thus, the comprehension of these regulatory mechanisms has become of major importance to disclose novel therapeutic targets. In this review, we first present a comprehensive view on the cell cycle components and mechanisms that were identified in the context of the homeostatic adult hippocampal neurogenic niche. Then, we focus on recent work regarding the cell cycle changes and signaling pathways that are responsible for the neurogenesis imbalances observed in neuropathological conditions, with a particular emphasis on depression.