G2-M Progression Research Articles

The Novel ATR Inhibitor M1774 Induces Replication Protein Overexpression and Broad Synergy with DNA-targeted Anticancer Drugs.

Ataxia telangiectasia and Rad3-related (ATR) checkpoint kinase inhibitors are in clinical trials. Here we explored the molecular pharmacology and therapeutic combination strategies of the oral ATR inhibitor M1774 (Tuvusertib) with DNA-damaging agents (DDA). As single agent, M1774 suppressed cancer cell viability at nanomolar concentrations, showing greater activity than ceralasertib and berzosertib, but less potency than gartisertib and elimusertib in the small cell lung cancer H146, H82, and DMS114 cell lines. M1774 also efficiently blocked the activation of the ATR-CHK1 checkpoint pathway caused by replication stress induced by TOP1 inhibitors. Combination with non-toxic dose of M1774 enhanced TOP1 inhibitor-induced cancer cell death by enabling unscheduled replication upon replicative damage, thereby increasing genome instability. Tandem mass tag-based quantitative proteomics uncovered that M1774, in the presence of DDA, forces the expression of proteins activating replication (CDC45) and G2-M progression (PLK1 and CCNB1). In particular, the fork protection complex proteins (TIMELESS and TIPIN) were enriched. Low dose of M1774 was found highly synergistic with a broad spectrum of clinical DDAs including TOP1 inhibitors (SN-38/irinotecan, topotecan, exatecan, and exatecan), the TOP2 inhibitor etoposide, cisplatin, the RNA polymerase II inhibitor lurbinectedin, and the PARP inhibitor talazoparib in various models including cancer cell lines, patient-derived organoids, and mouse xenograft models. Furthermore, we demonstrate that M1774 reverses chemoresistance to anticancer DDAs in cancer cells lacking SLFN11 expression, suggesting that SLFN11 can be utilized for patient selection in upcoming clinical trials.

SCFFBXW7 regulates G2-M progression through control of CCNL1 ubiquitination.

FBXW7, which encodes a substrate-specific receptor of an SCF E3 ligase complex, is a frequently mutated human tumor suppressor gene known to regulate the post-translational stability of various proteins involved in cellular proliferation. Here, using genome-wide CRISPR screens, we report a novel synthetic lethal genetic interaction between FBXW7 and CCNL1 and describe CCNL1 as a new substrate of the SCF-FBXW7 E3 ligase. Further analysis showed that the CCNL1-CDK11 complex is critical at the G2-M phase of the cell cycle since defective CCNL1 accumulation, resulting from FBXW7 mutation, leads to shorter mitotic time. Cells harboring FBXW7 loss-of-function mutations are hypersensitive to treatment with a CDK11 inhibitor, highlighting a genetic vulnerability that could be leveraged for cancer treatment.

Abstract 1971: RCC2 promotes glioblastoma through regulation of G2-M progression

Abstract Regulator of chromatin condensation 2 (RCC2), also known as telophase disk protein of 60 kDa (TD-60), has been previously identified as a component of the chromosome passenger complex. Accumulating evidence suggested that RCC2 plays a vital role in the mitotic process and abnormal RCC2 expression is essential for cancer development including Glioblastoma Multiforme (GBM). GBM is the most lethal and frequent brain tumor. Even with the optimal therapy, the 5-year survival rate of GBM remains poor. TCGA data revealed an upregulation of RCC2 in human GBM. Our preliminary data showed that RCC2 was upregulated in PTEN knockout mice primary astrocytes, and loss of function of PTEN has been found in nearly 95% GBM patients. This indicated that RCC2 might act as a PTEN downstream regulator and promote glioblastoma progression. RCC2 has been reported to be essential for tumorigenesis which may act as a GEF for Rac1 or RalA GTPase. Coincidently, our study showed that silencing RCC2 could suppress GBM cell proliferation. Using RNA-seq, we identified 737 DEGs between control siRNA- and RCC2 siRNA-treated A1172 cells. GSEA analysis delineated G2-M progression as the most notably altered pathway by silencing RCC2. Indeed, we observed a G2-M arrest in cells with RCC2 silenced, suggesting that RCC2 is essential for G2-M progression. Additionally, the connectivity map (C-map) analysis was conducted and identified LY294002 and cephaeline as promising inhibitors for RCC2-associated gene signatures. Both LY294002 and cephaeline can synergistically inhibit glioblastoma cell viability with RCC2 silencing. In summary, our study demonstrated that RCC2 signaling is essential for GBM development. We provided evidence that RCC2 exerts a positive effect on G2-M progression. These findings may shed light on anti-RCC2 targeted therapy in GBM treatment. Funding: This work is supported by a General Research Fund grant (#14113519) from the University Grants Committee of Hong Kong. Citation Format: Tian Liu, Penelope Mei-Yu Or, Chi-Wai Wong, Wayne Lut-Heng Ho, Stanley Kwok-Kuen Cheung, Yubing Wang, Andrew M. Chan. RCC2 promotes glioblastoma through regulation of G2-M progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1971.

Abstract 97: Analysis of Pten-null mouse astrocytes revealed overlapping transcribed genes with human glioblastoma transcriptome

Abstract Glioblastoma is an aggressive form of brain cancer with universally poor prognosis. Even with optimal therapy, the cancer recurs frequently. Phosphatase and tensin homolog (PTEN) is a tumor suppressor gene whose mutations are often found in multiple human cancers. PTEN encodes a lipid phosphatase and serves as a negative regulator of the phosphoinositide 3-kinase (PI3K) pathway. Cumulative evidence suggests that PTEN mutations play significant roles in tumorigenesis of glioblastoma as 60% of glioblastoma patients acquired mutations in this gene. Based on our preliminary data, transcriptomic profiling of primary astrocytes derived from Pten knockout mice revealed an overlapping enrichment of mitotic regulators and cell cycle related pathways with human GBM. 4 genes (RCC1, RCC2, NEK2, and CHK2) were validated to be significantly upregulated in PTEN KO astrocytes by qRT-PCR. Regulator of chromatin condensation 2 (RCC2) has been reported to be guanine-nucleotide exchange factor (GEF) for Rac1 and RalA GTPase and act as a regulator of cell cycle progression. Accumulating evidence revealed a pro-tumorigenic role of RCC2 in multiple cancers such as tumor growth and tumor metastasis. Via siRNA inhibition of RCC2, we demonstrated that knocking down of RCC2 in glioblastoma cell lines suppresses cell growth and RCC2 is essential for both G1-S and G2-M progression. Additionally, RCC2 knocking down and a PI3K inhibitor LY294002 inhibit glioblastoma cell viability in a synergistic manner. In summary, PTEN deficiency leads to the upregulation of RCC2, which may promote tumorigenesis of glioblastoma by dysregulating cell cycle progression.To further explore the RCC2 roles, we will find the potential downstream target of RCC2 in dysregulating cell cycle and its potential therapeutic effect in vivo. Funding: General Research Fund Grant #14113519 from The Research Grants Council of Hong Kong Citation Format: Tian Liu, Penelope Mei-Yu Or, Chi-Wai Wong, Stanley Kwok-Kuen Cheung, Yubing Wang, Yiwei Wang, Andrew M. Chan. Analysis of Pten-null mouse astrocytes revealed overlapping transcribed genes with human glioblastoma transcriptome [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 97.

E2F is required for STAT3-mediated upregulation of cyclin B1 and Cdc2 expressions and contributes to G2-M phase transition.

Activation of transcription factor STAT3 is involved in cell proliferation, differentiation, and cell survival. Constitutive activation of STAT3 pathway has been associated with the oncogenesis of various types of cancers. It has been reported that STAT3 plays a key role in the G1 to S phase cell cycle transition induced by the cytokine receptor subunit gp130, through the upregulation of cyclins D1, D2, D3, A, and Cdc25A and the concomitant downregulation of p21 and p27. However, its role in mediating G2-M phase transition has not been studied. The cyclin B1/Cdc2 complex is widely accepted as the trigger of mitosis in all organisms and is believed to be necessary for progression through S phase and keep active during the G2-M transition and progression. In the present study, we found that activation of STAT3 stimulates cyclin B1 and Cdc2 expressions. Deletion and site-directed mutations on cyclin B1 and Cdc2 promoters indicated that E2F element mediates the upregulation of these two promoters in a STAT3-dependent manner. The findings reported here demonstrated that STAT3 participates in modulating G2-M phase checkpoint by regulating gene expressions of cyclin B1 and Cdc2 via E2F.

X-linked α-thalassemia with mental retardation is downstream of protein kinase A in the meiotic cell cycle signaling cascade in Xenopus oocytes and is dynamically regulated in response to DNA damage†.

X-linked α-thalassemia with mental retardation (ATRX) is a chromatin remodeling protein that belongs to the SWItch/sucrose non-fermentable (SWI2/SNF2) family of helicase/ATPases. During meiosis, ATRX is necessary for heterochromatin formation and maintenance of chromosome stability in order to ensure proper assembly of the metaphase II spindle. Previously, we established ATRX as a novel progesterone regulated protein during bovine meiotic maturation, in addition to being dynamically regulated in response to DNA damage in oocytes. In the present study, we utilize the Xenopus laevis model system to further elucidate the signaling pathways regulating ATRX expression within the oocyte. Here, we present an analysis of endogenous ATRX protein expression during oogenesis, oocyte meiotic maturation, and early embryonic development. ATRX expression is dynamically regulated as evidenced by loss of the protein in metaphase II of meiosis. The downstream activation of meiosis via protein kinase A inhibition resulted in a similar decrease in ATRX protein expression. We demonstrate that the ATRX protein is detected in ubiquitin immuno-precipitates from germinal vesicle oocyte extracts and experimentally demonstrate that proteosomal degradation is responsible for the decreased expression of ATRX during meiosis. ATRX expression is significantly increased in response to gamma-irradiation induced DNA damage in oocytes and embryos. This increased expression is independent of p53 protein expression in apoptotic embryos, as determined by the expression of active caspase-3. Thus, regulation of ATRX protein expression impacts on G2-M progression and ultimately has consequences for cell survival.

The Chromosome 21 Kinase DYRK1A and Its Substrate FOXO1 Constitute a Novel Therapeutic Pathway in B-ALL

Dual Specificity Tyrosine-Phosphorylation-Regulated Kinase 1A (DYRK1A) is a serine/threonine kinase that regulates diverse pathways such as splicing, cell cycle, differentiation, apoptosis, and transcription. DYRK1A is encoded within the Down syndrome (DS) critical region of chromosome 21, underlying its importance in DS-related pathologies, such as Alzheimer's disease. Children with DS have an increased risk of developing hematologic malignancies, namely acute megakaryoblastic leukemia (DS-AMKL) and B-cell acute lymphoblastic leukemia (DS-ALL). We previously reported that DYRK1A promotes DS-AMKL by regulating subcellular localization of its substrate NFAT. In a subsequent study, we examined its role in normal hematopoiesis and found that DYRK1A is necessary for B and T cell development through phosphorylation and destabilization of Cyclin D3. Dyrk1a-deficient large pre-B cells and double negative thymocytes are unable to enter quiescence for maturation. Despite elevated levels of Cyclin D3, however, these cells lose proliferative capacity due to a block at the G2-M transition. This observation suggests that DYRK1A inhibition may exhibit anti-tumor activity in lymphocytes by first stimulating exit from quiescence but then blocking repeated rounds of cell division.Notably, DYRK1A is overexpressed in acute leukemias, including both T-ALL and B-ALL, relative to normal hematopoietic counterparts. Moreover, overexpression of dominant-negative DYRK1A-K188R impairs proliferation in human B-ALL cell lines, suggesting that DYRK1A kinase activity is required for B-ALL growth. In order to assess the physiologic relevance of targeting DYRK1A in vivo, we generated a murine model of B-ALL with a floxed Dyrk1a allele and observed significant survival advantages with homozygous (p=0.0045) and heterozygous deletion (p=0.0015). Additionally, both B-ALL cell lines and patient samples were sensitive to EHT1610, a potent and selective DYRK1 inhibitor. Relevant to the localization of DYRK1A on chromosome 21, DS-ALL samples were especially sensitive to kinase inhibition. EHT1610 also conferred synergistic growth inhibition of B-ALL cells when combined with cytotoxic chemotherapy drugs used in traditional ALL treatment regimens, such as dexamethasone, methotrexate and cytarabine.We next aimed to elucidate the mechanism by which DYRK1A inhibition cause a failure of G2-M progression. Using global and directed phosphoproteomic studies, we identified several DYRK1A substrates in pre-B cells that are involved in cell cycle, splicing, transcriptional regulation, and RNA metabolism. In addition to Cyclin D3, a notable substrate is FOXO1, an indispensable transcription factor in B lymphopoiesis. We observed that inhibition of DYRK1A led to an accumulation of FOXO1 in the nucleus of large pre-B cells despite intact PI3K/Akt signaling, which is the predominant negative regulator of FOXO1. Treatment of pre-B cells with AS1842856, an inhibitor of FOXO1 nuclear translocation, rescued the G2-M blockade and proliferative impairment induced by EHT1610 treatment. Despite FOXO1 acting as a tumor suppressor in normal lymphocytes, B-ALL cell lines and patient samples were paradoxically sensitive to FOXO1 inhibition, suggesting a unique requirement in the survival of B-ALL cells. This may be due to regulation of DNA damage, as DYRK1A inhibition alone led to negligible changes in gamma-H2AX foci, whereas FOXO1 inhibition increased DNA damage. When DYRK1A and FOXO1 were inhibited in combination, we observed a synergistic accumulation of DNA damage along with cell death in B-ALL cell lines.Finally, as both EHT1610 and AS1842856 are potent inhibitors of B-ALL cell growth in vitro, we assessed their in vivo efficacy. Both EHT1610 and AS1842856 significantly increased survival in xenograft models of B-ALL (p=0.0002 and p=0.001, respectively). We therefore conclude that both DYRK1A and its substrate FOXO1 are therapeutic targets in B-ALL. Importantly, EHT1610 represents the first selective DYRK1A inhibitor with suitable in vivo activity. Ultimately, we have determined that the DYRK1A pathway is integral to the maintenance of normal and malignant B-lymphopoiesis, the latter which can be effectively targeted through 1) a primary proliferative impairment, 2) sensitization to cell cycle-dependent chemotherapy, and 3) downstream inhibition of DYRK1A substrates such as FOXO1. DisclosuresLee:AbbVie: Employment. Bourquin:Amgen: Other: Travel Support. Crispino:Scholar Rock: Research Funding; Forma Therapeutics: Research Funding.

Abstract 4480: Irx1 gene knockout in zebrafish induces to multiorgan tumorigenesis through cell cycle aberration

Abstract The Iroquois homeobox 1 (IRX1) belongs to Iroquois homeobox family and plays an important role during embryonic development. Recent studies have suggested that IRX1 acts as a tumor suppressor in gastric cancer and head and neck carcinoma. The zebrafish allows easy genetic modification at a lower cost, thus being a valuable model for cancer biology. For functional annotation we established homozygous knockout mutants of Irx1a and Irx1b and followed to evaluate long-term phenotypes. Homozygous knockout zebrafish for either Irx1a or Irx1b are vital and survived long enough to see long-term phenotypes. However, majority of the homozygotes for both Irx1a /b were severely malformed, could not survive longer than 6 month, and were not fertile. Surprisingly, hyperplasia and tumorigenesis developed in homozygous Irx1a mutants in multiple organs, including bile duct, intestine, kidney, ovary, and testis where irx1 genes are expressed in adults when evaluated by in situ hybridization. Bile duct hyperplasia or tumor was the most common phenotype, occurring in half and two thirds at 6 and 12 months, respectively. Irx1b mutants also developed similar tumorigenesis but showed less prevalent phenotypes than Irx1a mutants, suggesting functional preservation and redundancy. Analysis of differential genes (Wild type vs. Irx1a/b knockout) by cRNA microarray (Agilent Zebrafish v3 GE 4X44 Microarrays) revealed 687 up- and 963 downregulated genes by Irx1a/b knockout. Significant canonical pathways were cyclins and cell cycle regulation, mitotic roles of polo-like kinase, FXR/RXR activation, estrogen-mediated S-phase entry, and cell cycle control of chromosomal replication, suggesting aberrant cell cycle regulation as an important mechanism of tumorigenesis. Real-time RT PCR validated the differential expression of cell cycle-related genes, especially genes involved in G2/M phase progression. Differential genes included CCND1, ARAF, CDKN2C, and FGFR1 among the upregulated genes, and ANAPC7, CCNA2, CCNB1, 2, HAUS1, 3, 6, CDC7, CDC20, CDCA8, FBXO5, 32, 43 and PLK3 among the downregulated genes. Overexpression by transfection decreased cell proliferation in cholangiocarcinoma cells (SNU-1196 and HuCCT1) without altering sensitivity to H2O2-induced apoptosis on annexin V flow cytometry. Western blot revealed decreased expression of CCND1, CCNE, CCNA1, CCNB1 by IRX1 expression. Flow cytometry using propidium iodide and Alexa647-anti-pHH3 showed decreased mitotic fraction by IRX1 expression. On cell cycle synchronization and release using hydroxyuria and nocodazole showed slight delay in G1-S progression and marked delay in G2-M progression and entry into G1. In conclusion, this study identified Irx1 gene as an important tumor-suppressor gene at various organs, and knockout or Irx1 leads to tumorigenesis by altering cell cycle regulation, especially at G2/M phase. Citation Format: Inhye Jung, Dawoon E. Jung, DoHee Kim, Dong Hee Kim, Yong Yoon Chung, Seung Woo Park. Irx1 gene knockout in zebrafish induces to multiorgan tumorigenesis through cell cycle aberration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4480.

FRA1 promotes squamous cell carcinoma growth and metastasis through distinct AKT and c-Jun dependent mechanisms

FRA1 (Fos-like antigen 1) is highly expressed in many epithelial cancers including squamous cell carcinoma of the skin (cSCC) and head and neck (HNSCC). However, the functional importance and the mechanisms mediating FRA1 function in these cancers are not fully understood. Here, we demonstrate that FRA1 gene silencing in HNSCC and cSCC cells resulted in two consequences – impaired cell proliferation and migration. FRA1 regulation of cell growth was distinct from that of c-Jun, a prominent Jun group AP-1 factor. While c-Jun was required for the expression of the G1/S phase cell cycle promoter CDK4, FRA1 was essential for AKT activation and AKT-dependent expression of CyclinB1, a molecule required for G2-M progression. Exogenous expression of a constitutively active form of AKT rescued cancer cell growth defect caused by FRA1-loss. Additionally, FRA1 knockdown markedly slowed cell adhesion and migration, and conversely expression of an active FRA1 mutant (FRA1DD) expedited these processes in a JNK/c-Jun-dependent manner. Through protein and ChIP-PCR analyses, we identified KIND1, a cytoskeletal regulator of the cell adhesion molecule β1-integrin, as a novel FRA1 transcriptional target. Restoring KIND1 expression rescued migratory defects induced by FRA1 loss. In agreement with these in vitro data, HNSCC cells with FRA1 loss displayed markedly reduced rates of subcutaneous tumor growth and pulmonary metastasis. Together, these results indicate that FRA1 promotes cancer growth through AKT, and enhances cancer cell migration through JNK/c-Jun, pinpointing FRA1 as a key integrator of JNK and AKT signaling pathways and a potential therapeutic target for cSCC and HNSCC.

Abstract 1991: FOXM1 expression in epithelial ovarian cancer coincides with gene amplification and functional loss of p53 and Rb and drives G2-M progression and target gene expression

Abstract Introduction: According to The Cancer Genome Atlas (TCGA), the FOXM1 transcription factor network is activated in 84% of high-grade serous ovarian cancers (HGS-OC), a common and lethal subtype of epithelial ovarian cancer (EOC). FOXM1 plays a key role in cell cycle progression and genomic stability, and is overexpressed in various malignancies. The goal of this study was to understand the genetic determinants of FOXM1 expression in EOC, and to define its functional role in relevant cell models. Methods: We analyzed FOXM1 gene amplification data in HGS-OC and other cancers using cBioPortal. We determined FOXM1 expression and its relationship to clinico-pathology in a large set of primary EOC tissues. We used primary and immortalized murine (MOSE) and human ovarian surface epithelial (hOSE) cells to determine the contribution of p53, Rb, and E2F1 to FOXM1 expression. We used FOXM1 knockdown and overexpression approaches to dissect its functional contribution to cell cycle progression and target gene activation in OSE and EOC cells. We used human EOC cell lines to assess genetic mechanisms driving the expression of the three known FOXM1 splice variants. Results: The FOXM1 locus was amplified in ∼12% of HGS-OC, greater than any other tumor type examined and suggestive of selective pressure in HGS-OC. FOXM1 mRNA was frequently upregulated in EOC, in association with advanced stage and grade. In MOSE cells, combined knockout of Rb1 and Trp53 synergistically induced Foxm1. In agreement, hOSE cells immortalized with SV40 T antigen (IOSE-SV) showed dramatically higher FOXM1 expression as compared to hOSE immortalized with hTERT. Knockdown experiments revealed that FOXM1 induction in IOSE-SV is partially dependent on E2F1. FOXM1 knockdown in IOSE-SV blocked G2-M transition and downregulated known targets of FOXM1, including CCNB1 and PLK1. Conversely, FOXM1 overexpression in primary hOSE upregulated FOXM1 target genes. FOXM1 expression in EOC cell lines was heterogeneous and the highest expression was observed in cell lines with FOXM1 gene amplification and TP53 and RB1 mutations. The level of expression of FOXM1 splice variants in EOC cell lines was consistently FOXM1c > FOXM1b > FOXM1a, indicating that transcriptionally active isoforms show higher expression. Conclusions: Our data reveal that genetic alterations in TP53 and RB1, along with FOXM1 gene amplification, are critical determinants of FOXM1 expression in EOC. FOXM1 functionally contributes to cell cycle progression in EOC models, likely via the transactivation of gene targets driving G2-M transition. Current studies are dissecting the role of post-translational modifications in FOXM1 activation in EOC, and extending work into relevant human fallopian tube secretory epithelial cell systems. Finally, our findings support the development of FOXM1-targeted therapies for EOC. Citation Format: Carter J. Barger, Wa Zhang, Kunle Odunsi, Adam R. Karpf. FOXM1 expression in epithelial ovarian cancer coincides with gene amplification and functional loss of p53 and Rb and drives G2-M progression and target gene expression. [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 1991. doi:10.1158/1538-7445.AM2015-1991

Abstract 1944: Divergent effects of BRAF activation in neural stem and progenitor-like glioblastoma cells

Abstract Glioblastoma multiforme (GBM) are heterogeneous tumors that are refractory to radiation and traditional chemotherapeutics. Separate studies have identified populations of neural stem and progenitor -like GBM cells, however the contribution of these cells types to tumor progression and the response to targeted therapy are poorly understood. Small molecules (such as vemurafenib) targeting the most common MAPK-activating mutation, BRAFV600E, are currently entering clinical trials for treatment of high-grade glioma. Understanding the contribution of different cell types to treatment responses will be necessary for the design of effective adjuvant therapeutic strategies. We show that activating MAPK pathway gene mutations in GBM correlate with elevated expression levels of oligodendrocyte precursor (OPC) and neural stem cell (NSC)-associated genes. Cells expressing CD133 and NG2 coexist in human GBM and BRafV600E expressing mouse neurosphere cultures, and maintain some of their respective neural stem and progenitor-like properties. In isogenic mouse cells expressing BRafV600E, NG2+ cells expand rapidly and symmetrically, while rates of asymmetric division are unaffected in slow-proliferating CD133+ cells. Tracing of proliferation rate with the membrane-associated dye PKH-26 reveals heterogeneity in the proliferation rate of CD133+ cells, but not proliferative NG2+ cells. BRAF-targeted inhibition of human GBM cells with PLX4720, the tool compound of vemurafenib, reduces S-phase entry of NG2+ cells, yet CD133+ cells are less affected. Interestingly targeted inhibition of components of the asymmetric division machinery selectively disrupts G2-M progression in CD133+ cells. The functionally divergent populations of CD133-expressing NSC-like cells will likely be therapeutically problematic with mono-therapeutic approaches. These data point to a differential regulation of key cell cycle processes between CD133+ stem-like cells and NG2+ OPC-like cells, that will inform the intelligent design of compound treatment strategies. Note: This abstract was not presented at the meeting. Citation Format: Robin G. Lerner, Yuichiro Ihara, Kate Lewis, Amelie Griveau, Brian Reichhold, Dian Qu, Martin McMahon, David Rowitch, Charles D. James, Claudia Petritsch. Divergent effects of BRAF activation in neural stem and progenitor-like glioblastoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1944. doi:10.1158/1538-7445.AM2014-1944

Abstract C217: Aneuploidy induction by the microtubule-stabilizing agent peloruside A involves the cell cycle checkpoint proteins MAD2 and p55CDC.

Abstract Peloruside A (PelA) is a novel secondary metabolite isolated from the New Zealand marine sponge Mycale hentscheli. It is a potent microtubule-stabilizer and binds to a distinct site on -tubulin compared to the widely used anti-cancer drug paclitaxel. PelA has clear potential benefits over paclitaxel, including increased solubility, reduced sensitivity to multiple drug resistance in cancer cell lines, and improved tolerability and efficacy in non-small cell lung cancer xenografts in mice. Previous chemical genetics studies have indicated that MAD2 (mitotic arrest deficiency-2), a spindle-assembly checkpoint (SAC) protein, could potentially be a secondary target of PelA. Premature dissociation of MAD2 from the SAC protein, p55CDC, has been implicated in the induction of aneuploidy in response to low concentrations of paclitaxel; thus, we sought to validate and investigate MAD2 as an additional target of PelA. Flow cytometric analysis of 1A9 (human ovarian carcinoma) cells confirmed that low concentrations of PelA (< 40 nM) induced chromosome mis-segregation in up to 15% of cells. Western blot analyses detected minimal changes in the abundance levels of MAD2, BUBR1 and p55CDC at both high (100 nM) and low (40 nM) PelA concentrations. However, differences in the association between MAD2 and BUBR1 with p55CDC were observed after cells were released from synchronization and assessed by co-immunoprecipitation. The mitotic arrest seen at 100 nM PelA was accompanied by a sustained formation of MAD2/p55CDC and BUBR1/p55CDC complexes; whereas, 40 nM PelA resulted in a similar dissociation profile of these complexes compared to controls. Given that PelA-induced aneuploidy coincides with G2-M progression, it is likely that dissociation of these complexes occurs despite aberrant microtubule-kinetochore attachments. Studies are currently underway to determine whether this increase in aneuploidy is due to the presence of erroneous attachments that are not detected by the SAC, such as merotelically oriented chromosomes. Aneuploidy is important because of its link to apoptosis and cancer cell death, and these experiments will help to elucidate some of the underlying signalling pathways. Additionally, these studies improve our understanding of the molecular actions of PelA during the cell cycle and highlight the importance of characterizing the concentration-dependent effects of drug interactions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C217.

P73 and p63 Sustain Cellular Growth by Transcriptional Activation of Cell Cycle Progression Genes

Despite extensive studies on the role of tumor suppressor p53 protein and its homologues, p73 and p63, following their overexpression or cellular stress, very little is known about the regulation of the three proteins in cells during physiologic cell cycle progression. We report a role for p73 and p63 in supporting cellular proliferation through the transcriptional activation of the genes involved in G(1)-S and G(2)-M progression. We found that in MCF-7 cells, p73 and p63, but not p53, are modulated during the cell cycle with a peak in S phase, and their silencing determines a significant suppression of proliferation compared with the control. Chromatin immunoprecipitation analysis shows that in cycling cells, p73 and p63 are bound to the p53-responsive elements (RE) present in the regulatory region of cell cycle progression genes. On the contrary, when the cells are arrested in G(0)-G(1), p73 detaches from the REs and it is replaced by p53, which represses the expression of these genes. When the cells move in S phase, p73 is recruited again and p53 is displaced or is weakly bound to the REs. These data open new possibilities for understanding the involvement of p73 and p63 in cancer. The elevated concentrations of p73 and p63 found in many cancers could cause the aberrant activation of cell growth progression genes and therefore contribute to cancer initiation or progression under certain conditions.

Potential role of estrogen receptor coregulator in mitosis.

Abstract Abstract #5036 Background: Estrogen stimulates breast tissue to increase cell divisions (mitosis) and is implicated in breast cancer progression. ER action is complex and requires functional interactions with coregulators. Proline-, glutamic acid-, and leucine-rich protein (PELP)-1, also known as modulator of nongenomic actions of estrogen receptor (MNAR), is a novel nuclear receptor (ER) coregulator with multitude of functions. Emerging evidence suggest that PELP1 expression is deregulated in breast cancer and serves as a scaffolding protein that couples various signaling complexes with estrogen receptor. In this study we found that ER coregulator PELP1 plays a novel role in mitosis.
 Material and Methods: To understand the mechanism by which ER coregulator PELP1 contribute to breast cancer progression, we have utilized small RNA interference methodology and established breast cancer model cells that stably express PELP1-shRNA (MCF7-PELP1shRNA). FACS analysis was used to determine the cell cycle status of the model cells. Using confocal microscopy, immunoprecipitation, in vitro kinase assays, site directed mutagenesis and Western analysis using phospho-antibodies we studied the mechanism and significance of PELP1 signaling in mitotic progression. We also developed PELP1 siRNA nanoparticles and used them as well as CDK1 inhibitors in cell proliferation studies.
 Results: Down regulation of PELP1 expression resulted in decreased estrogen mediated cell proliferation, delayed mitotic progression and induced accumulation of mitotic cells. Interestingly, PELP1 depleted cells also exhibited multinucleation. Western analysis of various markers of mitotic progression revealed a delay in the kinetics of G2M initiation and progression. Confocal analysis revealed colocalization of CDK1 and PELP1 in G2M. Immunoprecipitation assays demonstrate that endogenous CDK1 form functional complex with PELP1 and Src kinase during mitosis. Using deletion and mutagenesis approach, we have mapped the putative CDK1 phosphorylation sites on PELP1. Down regulation of PELP1 or overexpression of PELP1 mutants (that cannot be phosphorylated by CDK1), reduces the magnitude of Src activation, which is an essential driving force for timely progression of M phase. PELP1 siRNA nanoparticles alone or in combination with CDK1 inhibitors have shown to significantly reduce the proliferation of breast cancer cells and showed increased response in tamoxifen resistant breast cancer cells.
 Discussion: These results suggest that ER coregulator PELP1 play a novel role in G2M progression. Since PELP1 expression is deregulated in breast cancer, PELP1 ability to regulate mitosis could contribute to the progression of cancer by causing genomic instability through the deregulation of mitosis. Taken together our findings suggest that estrogen can promote neoplasia using coregulators by two distinct mechanisms (1) Coregulator induction of target genes and (2) Coregulator mediated actions in mitosis. ER coregulator PELP1 play multiple roles in Estrogen mediated neoplasia, and thus represent a target for novel therapeutic breast cancer strategies by forming the “next generation” of antimitotic drugs. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5036.

Nucleoplasmic mobilization of nucleostemin stabilizes MDM2 and promotes G2-M progression and cell survival

Nucleolar disassembly occurs during mitosis and nucleolar stress, releasing several MDM2-interactive proteins residing in the nucleolus that share the common activity of p53 stabilization. Here, we demonstrate that mobilization of nucleostemin, a nucleolar protein enriched in cancer and stem cells, has the opposite role of stabilizing MDM2 and suppressing p53 functions. Our results show that nucleostemin increases the protein stability and nucleoplasmic retention of MDM2, and competes with L23 for MDM2 binding. These activities were significantly elevated when nucleostemin is released into the nucleoplasm by mutations that abolish its nucleolar localization or by chemotherapeutic agents that disassemble the nucleoli. Nucleostemin depletion decreases MDM2 protein, increases transcription activity without affecting the level of p53 protein, and triggers G2-M arrest and cell death in U2OS cells but not in H1299 cells. This work reveals that nucleoplasmic relocation of nucleostemin during nucleolar disassembly safeguards the G2-M transit and survival of continuously dividing cells by MDM2 stabilization and p53 inhibition.

Human Immunodeficiency Virus Type 1 Vpr Binds to the N Lobe of the Wee1 Kinase Domain and Enhances Kinase Activity for Cdc2

Human immunodeficiency virus type 1 Vpr is a virion-associated accessory protein that has multiple activities within an infected cell. One of the most dramatic effects of Vpr is the induction of cell cycle arrest at the G(2)/M boundary, followed by apoptosis. This effect has implications for CD4(+) cell loss in AIDS. In normal cell cycle regulation, Wee1, a key regulator for G(2)-M progression, phosphorylates Tyr15 on Cdc2 and thereby blocks the progression of cells into M phase. We demonstrate that Vpr physically interacts with Wee1 at the N lobe of the kinase domain analogous to that present in other kinases. This interaction with Vpr enhances Wee1 kinase activity for Cdc2. Overexpression of Wee1 kinase-deficient mutants competes for Vpr-mediated cell cycle arrest, and deletion of the region of Wee1 that binds Vpr abrogates that competition. However, the Vpr mutants I74P and I81P, which fail to induce G(2) arrest, can bind to and increase the kinase activity of Wee1 to the same extent as wild-type Vpr. Therefore, we conclude that the binding of Vpr to Wee1 is not sufficient for Vpr to activate the G(2) checkpoint, and it may reflect an independent function of Vpr.

ROLE OF TETHERED ER MECHANISMS IN UTERINE RESPONSES

The estrogen receptor (ER) mediates the transcription of its target genes via two mechanisms. The classical mechanism involves direct interaction between the DNA binding domain of the ER and estrogen responsive enhancer (ERE) sequences in target genes. A second mechanism involves indirect tethered interaction between ER and other transcription factors such as AP1 which bind directly to DNA. To evaluate the role of the tethered mechanism in an in vivo model, the DNA binding domain of the ERalpha was inactivated by mutation and “knocked in” to a mouse. The resulting mouse, termed “Non Classical ER Knock In” (“NERKI”) could not be obtained as a homozygote, as the heterozygote was found to have severe female infertility due to ovarian and uterine pathologies. Heterozygous males were fertile and were mated to females heterozygous for the ERalpha null allele (alphaERKO) to produce offspring expressing the tethered selective NERKI allele as the only functional ERalpha (NERKI/alphaERKO). Although the NERKI/alphaERKO females showed no increase in uterine weight in response to estradiol (E2), epithelial cells increased in height and incorporated BrdU, indicating the tethered mechanism is sufficient for these epithelial responses. To evaluate the relative contributions of classical versus tethered mechanisms to the overall gene response profiles, microarrays of E2 treated WT and NERKI/alphaERKO uteri were compared. Results indicated selective preservation of E2 regulated uterine gene responses in the NERKI/alphaERKO. 1955 genes were regulated at least 2 fold by E2 at either 2 or 24 hours in aWT uterus, while only 407 genes were observed in the NERKI/alphaERKO. Interestingly, only 204 of these genes overlap in identity, indicating only about 10% of the WT uterine gene regulations (204/1955) remain in the NERKI/alphaERKO. In an in vitro culture system, Insulin-like growth factor 1 (Igf1) has previously been shown to be increased by E2 via ERalpha tethering with AP1. Igf1 transcript increased in the WT uterus with a maximal level 6 hours after E2 treatment (12.6 fold increase by RT-PCR). Therefore, we expected that Igf1 transcript would be induced by E2 in the NERKI/alphaERKO uterus. However, in the NERKI/alphaERKO uterus no increase in Igf1 transcript following E2 treatment was observed either by microarray analysis or by RT-PCR (1.3 fold increase), indicating that the tethered mechanism is not sufficient to regulate the endogenous Igf1 promoter in the mouse uterus. Our finding illustrates the potential for divergent regulation between certain in vitro model systems and in vivo models. Examination of the mouse Igf1 gene indicates a potential ERE sequence; the hypothesis that the mouse Igf1 gene is regulated by direct interaction with ER is under study. Additionally, previous reports demonstrated that E2 initiates G1-S progression but not G2-M progression in the uterine epithelium of the Igf1 null mouse; therefore we hypothesize that similar to the Igf1 KO, NERKI/alphaERKO epithelial cells undergo G1-S, as evidenced by BrdU incorporation, but as the NERKI/alphaERKO uterus lacks any E2- mediated increase in Igf1, the epithelial cells will not progress through G2-M. To assess this hypothesis the induction of mitosis by E2 in the NERKI/alphaERKO uterine epithelium is being evaluated. (platform)

Robert H. Costa: In Memoriam (1957-2006)

Robert Henry (Rob) Costa, an authority on liver biology and cancer, died on September 1, 2006 at his home in Oak Park, IL. Rob was born in Galesburg, IL and grew up in Bethesda, MD. He attended the University of California at Irvine, where he received his doctorate in Molecular Virology in 1985 under the guidance of Dr. Edward K. Wagner. Rob characterized several genes encoded by the type I herpes simplex virus at a time when many techniques of molecular biology were still in their infancy. Rob was instrumental in identifying the viral transcripts, including the late genes, which are important in the viral life cycle.In 1985, Rob joined the laboratory of Dr. James E. Darnell, Jr. at the Rockefeller University for postdoctoral training. At that time, the Darnell laboratory was testing the hypothesis that tissue-enriched transcription factors were responsible for tissue-specific gene expression and tissue development. Rob, along with two other colleagues, Drs. Eseng Lai and Dennis R. Grayson, characterized the promoter region of the mouse prealbumin gene, which is mainly expressed in the liver. More importantly, Rob and colleagues discovered and characterized several liver-enriched transcription factors, including HNF3s or FoxAs, some of which were later shown to be also critical for mammalian development. These studies provided insights into mechanisms for liver-specific gene expression and laid the foundation of our current understanding of liver development.Rob joined the Northwestern University Medical School as an Assistant Professor in 1988. In 1989, he moved to the University of Illinois at Chicago where he rose to the rank of professor of biochemistry and molecular genetics. Throughout his career, Rob believed in digging deep for new information with cutting edge technologies. He was an imaginative thinker and was passionate about the process of discovery. He would leave the comfort zone and apply novel approaches much more easily than many of us. He was always generous with his time, reagents, and thoughtful advice. He exuded energy and enthusiasm, making his presence felt in a very positive way.Rob directed a vigorous research program. His laboratory identified a number of the fork-head family of genes that play key roles in liver-specific gene expression. Among his many contributions is the discovery of FoxM1b, a critical gene that is essential for liver development and liver cell proliferation. He showed that increasing the expression of FoxM1b in liver cells of aged mice make them behave as though they were cells in young mice, thereby restoring the proliferation potential of juveniles in aged animals. This work was published in the Proceedings of the National Academy of Science in 2002 and featured in ASBMB Today in the February, 2003 issue. His laboratory showed that FoxM1b plays a crucial role in mammalian cell division by stimulating expression of genes necessary for cell cycle progression. Several genes that regulate G2-M progression are critically dependent on FoxM1b, and cells lacking FoxM1b fail to enter M phase.More recently, Rob's laboratory showed that expression of FoxM1b is essential for the development of liver cancer. Conditional deletion of FoxM1b in the liver rendered mice refractory to hepatocellular carcinoma (HCC) development following treatment with DEN/Phenobarbital, which causes HCC in littermates expressing normal level of FoxM1b. His laboratory also discovered that FoxM1b is a target of the tumor suppressor ARF. This work was published in Genes & Development in 2004. Based on these studies, he pioneered a new approach to treat liver cancer by using small molecules derived from ARF to disrupt the function of FoxM1b. This new approach is currently under development.Widely regarded as a star in the liver field, Rob was a leading authority in the molecular analysis of liver biology and cell proliferation. He organized several prestigious national conferences on liver gene expression and development and chaired scientific sessions at major international conferences in the United States and Europe. He served as an editorial board member and peer reviewer for several prestigious journals, including Hepatology, The Journal Biological Chemistry and several of the AACR journals. In addition, Rob sat on numerous review panels for the NIH.A respected innovator and beloved colleague, Rob trained numerous graduate students and postdocs and helped mentor many junior faculty members. In February, 2006, he was diagnosed with stage IV pancreatic cancer with liver and bone metastases. Rob battled his illness heroically with uncommon courage, grace, and fortitude. He never gave up hope or his sense of humor, and he continued to work assiduously while undergoing chemotherapy. His wife Jane and son Sam, the anchors of his life, made it possible for him to stay optimistic. His death at the height of his career is a tragic loss to science and the scientific community. In him, we will remember an outstanding scientist, a loyal friend, an exemplary mentor, and a fine human being. We will miss him greatly.

Combined Depletion of Cell Cycle and Transcriptional Cyclin-Dependent Kinase Activities Induces Apoptosis in Cancer Cells

Selective cyclin-dependent kinase (cdk) 2 inhibition is readily compensated. However, reduced cdk2 activity may have antiproliferative effects in concert with other family members. Here, inducible RNA interference was used to codeplete cdk2 and cdk1 from NCI-H1299 non-small cell lung cancer and U2OS osteosarcoma cells, and effects were compared with those mediated by depletion of either cdk alone. Depletion of cdk2 slowed G1 progression of NCI-H1299 cells and depletion of cdk1 slowed G2-M progression in both cell lines, with associated endoreduplication in U2OS cells. However, compared with the incomplete cell cycle blocks produced by individual depletion, combined depletion had substantial consequences, with G2-M arrest predominating in NCI-H1299 cells and apoptosis the primary outcome in U2OS cells. In U2OS cells, combined depletion affected RNA polymerase II expression and phosphorylation, causing decreased expression of the antiapoptotic proteins Mcl-1 and X-linked inhibitor of apoptosis (XIAP), effects usually mediated by inhibition of the transcriptional cdk9. These events do not occur after individual depletion of cdk2 and cdk1, suggesting that reduction of cdk2, cdk1, and RNA polymerase II activities all contribute to apoptosis in U2OS cells. The limited cell death induced by combined depletion in NCI-H1299 cells was significantly increased by codepletion of cdk9 or XIAP or by simultaneous treatment with the cdk9 inhibitor flavopiridol. These results show the potency of concomitant compromise of cell cycle and transcriptional cdk activities and may guide the selection of clinical drug candidates.

Repression of cell cycle–related proteins by oxaliplatin but not cisplatin in human colon cancer cells

Oxaliplatin (Eloxatin) is a third-generation platinum derivative with an in vitro and in vivo spectrum of activity distinct from that of cisplatin, especially in colon cancer cells. Here, we studied the molecular basis of this difference on the HCT-116 human colon carcinoma cell line (mismatch repair-deficient, wild-type functional p53). Oxaliplatin inhibited HCT-116 cell proliferation with greater efficacy than cisplatin. At comparable concentrations, cisplatin slowed down the replication phase and activated the G2-M checkpoint, whereas oxaliplatin activated the G1-S checkpoint and completely blocked the G2-M transition. With the aim of finding oxaliplatin-specific target genes and mechanisms differing from those of cisplatin, we established the transcriptional signatures of both products on HCT-116 cells using microarray technology. Based on hierarchical clustering, we found that (a) many more genes were modulated by oxaliplatin compared with cisplatin and (b) among the 117 modulated genes, 79 were regulated similarly by both drugs and, in sharp contrast, 38 genes were dose dependently down-regulated by oxaliplatin and, conversely, up-regulated or unaffected by cisplatin. Interestingly, several cell cycle-related genes encoding proteins involved in DNA replication and G2-M progression belong to this latter group. RNA modulations, confirmed at the protein level, were in accordance with oxaliplatin- and cisplatin-induced cell cycle variations. Beyond the identification of genes affected by both drugs, the identified oxaliplatin-specific target genes could be useful as predictive markers for evaluating and comparing the efficacy and molecular pharmacology of platinum drugs.