Cell Cycle Progression In Response Research Articles

Interdependency of estradiol-mediated ERα activation and subsequent PR and GREB1 induction to control cell cycle progression

Various groups of chemicals that we encounter in every-day life are known to disrupt the endocrine system, such as estrogen mimics that can disturb normal cellular development and homeostasis. To understand the effect of estrogen on intracellular protein dynamics and how this relates to cell proliferation, we aimed to develop a quantitative description of transcription factor complexes and their regulation of cell cycle progression in response to estrogenic stimulation. We designed a mathematical model that describes the dynamics of three proteins, GREB1, PR and TFF1, that are transcriptionally activated upon binding of 17β-estradiol (E2) to estrogen receptor alpha (ERα). Calibration of this model to imaging data monitoring the expression dynamics of these proteins in MCF7 cells suggests that transcriptional activation of GREB1 and PR depends on the association of the E2-ERα complex with both GREB1 and PR. We subsequently combined this ER signaling model with a previously published cell cycle model and compared this to quantification of cell cycle durations in MCF7 cells following nuclei tracking based on images segmented with deep neural networks. The resulting model predicts the effect of GREB1 and PR knockdown on cell cycle progression, thus providing mechanistic insight in the molecular interactions between ERα-regulated proteins and their relation to cell cycle progression. Our findings form a valuable basis to further investigate the pharmacodynamics of endocrine disrupting chemicals and their influence on cellular behavior.

Quantifying cell cycle regulation by tissue crowding

The spatiotemporal coordination and regulation of cell proliferation is fundamental in many aspects of development and tissue maintenance. Cells have the ability to adapt their division rates in response to mechanical constraints, yet we do not fully understand how cell proliferation regulation impacts cell migration phenomena. Here, we present a minimal continuum model of cell migration with cell cycle dynamics, which includes density-dependent effects and hence can account for cell proliferation regulation. By combining minimal mathematical modeling, Bayesian inference, and recent experimental data, we quantify the impact of tissue crowding across different cell cycle stages in epithelial tissue expansion experiments. Our model suggests that cells sense local density and adapt cell cycle progression in response, during G1 and the combined S/G2/M phases, providing an explicit relationship between each cell-cycle-stage duration and local tissue density, which is consistent with several experimental observations. Finally, we compare our mathematical model’s predictions to different experiments studying cell cycle regulation and present a quantitative analysis on the impact of density-dependent regulation on cell migration patterns. Our work presents a systematic approach for investigating and analyzing cell cycle data, providing mechanistic insights into how individual cells regulate proliferation, based on population-based experimental measurements.

Abnormal expression of cortical cell cycle regulators underlying anxiety and depressive-like behavior in mice exposed to chronic stress.

The cell cycle is a critical mechanism for proper cellular growth, development and viability. The p16INK4a and p21Waf1/Cip1 are important regulators of the cell cycle progression in response to internal and external stimuli (e.g., stress). Accumulating evidence indicates that the prefrontal cortex (PFC) is particularly vulnerable to stress, where stress induces, among others, molecular and morphological alterations, reflecting behavioral changes. Here, we investigated if the p16INK4a and p21Waf1/Cip1 expression are associated with behavioral outcomes. Prefrontal cortex mRNA and protein levels of p16INK4A and p21Waf1/Cip1 of mice (six independent groups of C57BL/6J, eight mice/group, 50% female) exposed from 0 to 35 days of chronic restraint stress (CRS) were quantified by qPCR and Western Blot, respectively. Correlation analyses were used to investigate the associations between cyclin-dependent kinase inhibitors (CKIs) expression and anxiety- and depression-like behaviors. Our results showed that the PFC activated the cell cycle regulation pathways mediated by both CKIs p16INK4A and p21Waf1/Cip1 in mice exposed to CRS, with overall decreased mRNA expression and increased protein expression. Moreover, correlation analysis revealed that mRNA and protein levels are statistically significant correlated with anxiety and depressive-like behavior showing a greater effect in males than females. Our present study extends the existing literature providing evidence that PFC cells respond to chronic stress exposure by overexpressing CKIs. Furthermore, our findings indicated that abnormal expression of p16INK4A and p21Waf1/Cip1 may significantly contribute to non-adaptive behavioral responses.

The SKP2-p27 axis defines susceptibility to cell death upon CHK1 inhibition.

Checkpoint kinase 1 (CHK1; encoded by CHEK1) is an essential gene that monitors DNA replication fidelity and prevents mitotic entry in the presence of under‐replicated DNA or exogenous DNA damage. Cancer cells deficient in p53 tumor suppressor function reportedly develop a strong dependency on CHK1 for proper cell cycle progression and maintenance of genome integrity, sparking interest in developing kinase inhibitors. Pharmacological inhibition of CHK1 triggers B‐Cell CLL/Lymphoma 2 (BCL2)‐regulated cell death in malignant cells largely independently of p53, and has been suggested to kill p53‐deficient cancer cells even more effectively. Next to p53 status, our knowledge about factors predicting cancer cell responsiveness to CHK1 inhibitors is limited. Here, we conducted a genome‐wide CRISPR/Cas9‐based loss‐of‐function screen to identify genes defining sensitivity to chemical CHK1 inhibitors. Next to the proapoptotic BCL2 family member, BCL2 Binding Component 3 (BBC3; also known as PUMA), the F‐box protein S‐phase Kinase‐Associated Protein 2 (SKP2) was validated to tune the cellular response to CHK1 inhibition. SKP2 is best known for degradation of the Cyclin‐dependent Kinase Inhibitor 1B (CDKN1B; also known as p27), thereby promoting G1‐S transition and cell cycle progression in response to mitogens. Loss of SKP2 resulted in the predicted increase in p27 protein levels, coinciding with reduced DNA damage upon CHK1‐inhibitor treatment and reduced cell death in S‐phase. Conversely, overexpression of SKP2, which consequently results in reduced p27 protein levels, enhanced cell death susceptibility to CHK1 inhibition. We propose that assessing SKP2 and p27 expression levels in human malignancies will help to predict the responsiveness to CHK1‐inhibitor treatment.

The N-Terminal Region of the Polo Kinase Cdc5 Is Required for Downregulation of the Meiotic Recombination Checkpoint.

During meiosis, the budding yeast polo-like kinase Cdc5 is a crucial driver of the prophase I to meiosis I (G2/M) transition. The meiotic recombination checkpoint restrains cell cycle progression in response to defective recombination to ensure proper distribution of intact chromosomes to the gametes. This checkpoint detects unrepaired DSBs and initiates a signaling cascade that ultimately inhibits Ndt80, a transcription factor required for CDC5 gene expression. Previous work revealed that overexpression of CDC5 partially alleviates the checkpoint-imposed meiotic delay in the synaptonemal complex-defective zip1Δ mutant. Here, we show that overproduction of a Cdc5 version (Cdc5-ΔN70), lacking the N-terminal region required for targeted degradation of the protein by the APC/C complex, fails to relieve the zip1Δ-induced meiotic delay, despite being more stable and reaching increased protein levels. However, precise mutation of the consensus motifs for APC/C recognition (D-boxes and KEN) has no effect on Cdc5 stability or function during meiosis. Compared to the zip1Δ single mutant, the zip1Δ cdc5-ΔN70 double mutant exhibits an exacerbated meiotic block and reduced levels of Ndt80 consistent with persistent checkpoint activity. Finally, using a CDC5-inducible system, we demonstrate that the N-terminal region of Cdc5 is essential for its checkpoint erasing function. Thus, our results unveil an additional layer of regulation of polo-like kinase function in meiotic cell cycle control.

HIF1\u03b1/HIF2\u03b1\u2013Sox2/Klf4 promotes the malignant progression of glioblastoma via the EGFR\u2013PI3K/AKT signalling pathway with positive feedback under hypoxia

Previous studies have suggested that hypoxic responses are regulated by hypoxia-inducible factors (HIFs), which in turn promote the malignant progression of glioblastoma (GBM) by inhibiting apoptosis and increasing proliferation; these events lead to a poor prognosis of GBM patients. However, there are still no HIF-targeted therapies for the treatment of GBM. We have conducted series of experiments and discovered that GBM cells exhibit features indicative of malignant progression and are present in a hypoxic environment. Knocking out HIF1α or HIF2α alone resulted in no significant change in cell proliferation and cell cycle progression in response to acute hypoxia, but cells showed inhibition of stemness expression and chemosensitization to temozolomide (TMZ) treatment. However, simultaneously knocking out HIF1α and HIF2α inhibited cell cycle arrest and promoted proliferation with decreased stemness, making GBM cells more sensitive to chemotherapy, which could improve patient prognosis. Thus, HIF1α and HIF2α regulate each other with negative feedback. In addition, HIF1α and HIF2α are upstream regulators of epidermal growth factor (EGF), which controls the malignant development of GBM through the EGFR–PI3K/AKT–mTOR–HIF1α signalling pathway. In brief, the HIF1α/HIF2α–EGF/EGFR–PI3K/AKT–mTOR–HIF1α signalling axis contributes to the growth of GBM through a positive feedback mechanism. Finally, HIF1α and HIF2α regulate Sox2 and Klf4, contributing to stemness expression and inducing cell cycle arrest, thus increasing malignancy in GBM. In summary, HIF1α and HIF2α regulate glioblastoma malignant progression through the EGFR–PI3K/AKT pathway via a positive feedback mechanism under the effects of Sox2 and Klf4, which provides a new tumour development model and strategy for glioblastoma treatment.

The MDM2 ligand Nutlin-3 differentially alters expression of the immune blockade receptors PD-L1 and CD276

BackgroundThe links between the p53/MDM2 pathway and the expression of pro-oncogenic immune inhibitory receptors in tumor cells are undefined. In this report, we evaluate whether there is p53 and/or MDM2 dependence in the expression of two key immune receptors, CD276 and PD-L1.MethodsProximity ligation assays were used to quantify protein-protein interactions in situ in response to Nutlin-3. A panel of p53-null melanoma cells was created using CRISPR-Cas9 guide RNA mediated genetic ablation. Flow cytometric analyses were used to assess the impact of TP53 or ATG5 gene ablation, as well as the effects of Nutlin-3 and an ATM inhibitor on cell surface PD-L1 and CD276. Targeted siRNA was used to deplete CD276 to assess changes in cell cycle parameters by flow cytometry. A T-cell proliferation assay was used to assess activity of CD4+ T-cells as a function of ATG5 genotype.ResultsCD276 forms protein-protein interactions with MDM2 in response to Nutlin-3, similar to the known MDM2 interactors p53 and HSP70. Isogenic HCT116 p53-wt/null cancer cells demonstrated that CD276 is induced on the cell surface by Nutlin-3 in a p53-dependent manner. PD-L1 was also unexpectedly induced by Nutlin-3, but PD-L1 does not bind MDM2. The ATM inhibitor KU55993 reduced the levels of PD-L1 under conditions where Nutlin-3 induces PD-L1, indicating that MDM2 and ATM have opposing effects on PD-L1 steady-state levels. PD-L1 is also up-regulated in response to genetic ablation of TP53 in A375 melanoma cell clones under conditions in which CD276 remains unaffected. A549 cells with a deletion in the ATG5 gene up-regulated only PD-L1, further indicating that PD-L1 and CD276 are under distinct genetic control.ConclusionGenetic inactivation of TP53, or the use of the MDM2 ligand Nutlin-3, alters the expression of the immune blockade receptors PD-L1 and CD276. The biological function of elevated CD276 is to promote altered cell cycle progression in response to Nutlin-3, whilst the major effect of elevated PD-L1 is T-cell suppression. These data indicate that TP53 gene status, ATM and MDM2 influence PD-L1 and CD276 paralogs on the cell surface. These data have implications for the use of drugs that target the p53 pathway as modifiers of immune checkpoint receptor expression.

The type IV pilin PilA couples surface attachment and cell-cycle initiation in Caulobacter crescentus

Understanding how bacteria colonize surfaces and regulate cell-cycle progression in response to cellular adhesion is of fundamental importance. Here, we use transposon sequencing in conjunction with fluorescence resonance energy transfer (FRET) microscopy to uncover the molecular mechanism for how surface sensing drives cell-cycle initiation in Caulobacter crescentus We identify the type IV pilin protein PilA as the primary signaling input that couples surface contact to cell-cycle initiation via the second messenger cyclic di-GMP (c-di-GMP). Upon retraction of pili filaments, the monomeric pilin reservoir in the inner membrane is sensed by the 17-amino acid transmembrane helix of PilA to activate the PleC-PleD two-component signaling system, increase cellular c-di-GMP levels, and signal the onset of the cell cycle. We termed the PilA signaling sequence CIP for "cell-cycle initiating pilin" peptide. Addition of the chemically synthesized CIP peptide initiates cell-cycle progression and simultaneously inhibits surface attachment. The broad conservation of the type IV pili and their importance in pathogens for host colonization suggests that CIP peptide mimetics offer strategies to inhibit surface sensing, prevent biofilm formation and control persistent infections.

Nuclear envelope deformation controls cell cycle progression in response to mechanical force.

The shape of the cell nucleus can vary considerably during developmental and pathological processes; however, the impact of nuclear morphology on cell behavior is not known. Here, we observed that the nuclear envelope flattens as cells transit from G1 to S phase and inhibition of myosin II prevents nuclear flattening and impedes progression to S phase. Strikingly, we show that applying compressive force on the nucleus in the absence of myosin II‐mediated tension is sufficient to restore G1 to S transition. Using a combination of tools to manipulate nuclear morphology, we observed that nuclear flattening activates a subset of transcription factors, including TEAD and AP1, leading to transcriptional induction of target genes that promote G1 to S transition. In addition, we found that nuclear flattening mediates TEAD and AP1 activation in response to ROCK‐generated contractility or cell spreading. Our results reveal that the nuclear envelope can operate as a mechanical sensor whose deformation controls cell growth in response to tension.

Abstract 4647: Keratinocyte specific deletion of TWIST1 promotes differentiation and inhibits UVB-induced hyperproliferation

Abstract Non-melanoma skin cancers (NMSC) have one of the highest incidences in the United States with over 5 million diagnoses every year. The major risk factor for the development of NMSC is solar ultraviolet radiation consisting of both UVA and UVB. In epidermal keratinocytes, UV radiation results in DNA damage as well as deregulation of both proliferative and survival signaling pathways that contribute to tumorigenesis. The transcription factor TWIST1 has been reported to be essential for the formation and invasiveness of chemically-induced tumors in skin. TWIST1 deletion in mouse epidermis results in arrested cell cycle progression in response to treatment with the tumor promoter, TPA, which in turn prevents skin tumor growth in two-stage skin carcinogenesis assays. However, the impact of the keratinocyte specific TWIST1 deletion on skin carcinogenesis caused by UV radiation has not been clarified. In preliminary experiments, we found that primary mouse keratinocytes with TWIST1 knockout (KO) displayed an increase in the G2/M phase population after a single exposure to 20mJ/cm2 of UVB radiation. Accordingly, protein levels of cell cycle regulator p21 increased while those of pMDM2 decreased after UVB exposure. Moreover, we found that primary mouse keratinocytes with TWIST1 KO displayed an increase in apoptosis as shown by augmented Annexin V staining that corresponded with the reduction in levels of anti-apoptotic regulators Bcl-2 and Bcl-XL and concomitant upregulation of cleaved PARP and cleaved caspase-7 after UVB exposure. Furthermore, deletion of TWIST1 in skin keratinocytes in vivo (using K5-Cre x TWIST1flox/flox mice) led to a significant reduction in UVB-induced epidermal hyperproliferation compared to wild-type mice assessed by both Ki67 immunofluorescence staining as well as BrdU incorporation. Additionally, the protein level for p21 was increased while the levels of c-myc, and Cyclin B1 were reduced in the TWIST1 deficient keratinocytes in vivo. Interestingly, expression of differentiation markers such as Loricrin, Keratin-10, Sca-1, and transglutaminase-1 were also increased in the epidermis of TWIST1 deficient mice. These results from the in vivo model show that TWIST1 deletion results in an interruption of UVB-induced hyperproliferation via cell cycle arrest and induction of keratinocyte differentiation. Collectively, our findings indicate that the deficiency of TWIST1 has significant effects on the proliferation, survival and differentiation of keratinocytes; processes important for UVB skin tumor development. Future experiments will continue to assess the changes in oncogenic signaling in epidermal keratinocytes in response to UVB exposure and further investigate the impact of the deletion of TWIST1 on UV-induced skin carcinogenesis. Research supported by CPRIT 2018 Grant RP150247 and by CONACYT Fellowship 671661. Citation Format: Fernando Eguiarte-Solomon, Okkyung Rho, John DiGiovanni. Keratinocyte specific deletion of TWIST1 promotes differentiation and inhibits UVB-induced hyperproliferation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4647.

Abstract 2551: Inhibition of mTOR abrogates mitosis progression

Abstract The mammalian target of rapamycin (mTOR) mediates the coordination of cell growth and cell cycle progression in response to nutrient supply. It is frequently activated in cancers and confers resistance to anti-cancer drugs. Accumulated studies have demonstrated that inhibition of mitotic kinases (PLK1 or aurora kinase A) enhances mTOR activities and the mTOR activation is associated with cell survival to anti-mitotic drugs. In this study, whether mTOR signaling is involved in mitosis progression is explored. Treatment of cells with mTOR inhibitors for 24 h resulted in the accumulation of G2 and mitotic cells and increased the percentage of mitotic cells with spindle abnormalities and chromosome missegregation. In addition, mTOR inhibitors sensitized cancer cells to taxol by enhancing apoptosis. Our results indicate that mTOR signaling pathway may be required for faithful mitosis progression and represent a potential resistance mechanism to the use of anti-mitotic drugs. Citation Format: Hsiao-Hui Kuo, Chieh-Ting Fang, Ling-Huei Yih. Inhibition of mTOR abrogates mitosis progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2551.

Characterization of Pch2 localization determinants reveals a nucleolar-independent role in the meiotic recombination checkpoint.

The meiotic recombination checkpoint blocks meiotic cell cycle progression in response to synapsis and/or recombination defects to prevent aberrant chromosome segregation. The evolutionarily conserved budding yeast Pch2TRIP13 AAA+ ATPase participates in this pathway by supporting phosphorylation of the Hop1HORMAD adaptor at T318. In the wild type, Pch2 localizes to synapsed chromosomes and to the unsynapsed rDNA region (nucleolus), excluding Hop1. In contrast, in synaptonemal complex (SC)-defective zip1Δ mutants, which undergo checkpoint activation, Pch2 is detected only on the nucleolus. Alterations in some epigenetic marks that lead to Pch2 dispersion from the nucleolus suppress zip1Δ-induced checkpoint arrest. These observations have led to the notion that Pch2 nucleolar localization could be important for the meiotic recombination checkpoint. Here we investigate how Pch2 chromosomal distribution impacts checkpoint function. We have generated and characterized several mutations that alter Pch2 localization pattern resulting in aberrant Hop1 distribution and compromised meiotic checkpoint response. Besides the AAA+ signature, we have identified a basic motif in the extended N-terminal domain critical for Pch2's checkpoint function and localization. We have also examined the functional relevance of the described Orc1-Pch2 interaction. Both proteins colocalize in the rDNA, and Orc1 depletion during meiotic prophase prevents Pch2 targeting to the rDNA allowing unwanted Hop1 accumulation on this region. However, Pch2 association with SC components remains intact in the absence of Orc1. We finally show that checkpoint activation is not affected by the lack of Orc1 demonstrating that, in contrast to previous hypotheses, nucleolar localization of Pch2 is actually dispensable for the meiotic checkpoint.

Headcase and Unkempt Regulate Tissue Growth and Cell Cycle Progression in Response to Nutrient Restriction

SUMMARYNutrient restriction (NR) decreases the incidence and growth of many types of tumors, yet the underlying mechanisms are not fully understood. In this study, we identified Headcase (Hdc) and Unkempt (Unk) as two NR-specific tumor suppressor proteins that form a complex to restrict cell cycle progression and tissue growth in response to NR in Drosophila. Loss of Hdc or Unk does not confer apparent growth advantage under normal nutrient conditions but leads to accelerated cell cycle progression and tissue overgrowth under NR. Hdc and Unk bind to the TORC1 component Raptor and preferentially regulate S6 phosphorylation in a TORC1-dependent manner. We further show that HECA and UNK, the human counterparts of Drosophila Hdc and Unk, respectively, have a conserved function in regulating S6 phosphorylation and tissue growth. The identification of Hdc and Unk as two NR-specific tumor suppressors provides insight into molecular mechanisms underlying the anti-tumorigenic effects of NR.

P53 Activates the Long Noncoding RNA Pvt1b to Inhibit Myc and Suppress Tumorigenesis

The p53 pathway is a key tumor suppressor mechanism that stalls cell cycle progression in response to stress. It has been proposed that p53 suppresses proliferation in part by downregulating the Myc oncogene but the mechanism has remained unclear. Here, we show that p53 directly represses Myc through a long noncoding RNA expressed from the Pvt1 locus. Pvt1b, a p53-dependent RNA isoform, is rapidly induced upon genotoxic and oncogenic stress and accumulates at its site of transcription in foci that exhibit properties of liquid-phase condensates. Pvt1b uses long-range chromatin interactions between Pvt1b and Myc to locally suppress Myc in an RNA-dependent manner. Loss of p53-dependent Pvt1b induction leads to increased cellular proliferation in vitro and enhanced tumor growth in a mouse model of lung adenocarcinoma in vivo. We propose that Pvt1b-mediated Myc repression contributes to the p53 tumor suppressive network to limit cancer growth.

Abstract LB-B13: Pediatric Preclinical Testing Consortium evaluation of the CHK1 inhibitor prexasertib

Abstract Introduction: Prexasertib is a CHK1 inhibitor that has entered clinical evaluation in adults and children with cancer. CHK1 plays a central role in pausing cell cycle progression in response to DNA damage, and an RNAi screen identified CHK1 as a therapeutic target for neuroblastoma (Cole KA, PNAS 2011; 108: 3336-41). Methods: Prexasertib was tested in vitro against a 23 cell line panel at concentrations from 0.1 nM to 1 µM. In vivo treatment groups included: prexasertib at 7.5 and 10 mg/kg given BID on days 1-3 x 3 weeks (Regimen [Reg] F & B, respectively); prexasertib at 4 mg/kg given BID days 1, 3, 5 (Reg C); irinotecan (IR) at 2.5 mg/kg days 1-5 (Reg D); and the combination of the agents (Reg E) with prexasertib as per Reg C and IR as per Reg D. For solid tumor testing, events are defined as 4X increase in tumor volume from day 0 and for CNS tumor testing as animals becoming moribund or developing severe neurologic deficit. The Kaplan-Meier method was used to compare EFS between treated and control groups. The objective response categories are progressive disease (PD, which is subdivided into progressive disease without and with growth delay, PD1 and PD2 respectively), stable disease (SD), partial response (PR), complete response (CR) and maintained complete response (MCR) (Houghton PJ, et al. Pediatr Blood Cancer 2007;49:928-40). Prexasertib was provided for testing by Lilly. Results: The prexasertib median IC50 for the pediatric cell lines was 3.2 nM, with a range from 0.9 nM to 22 nM and with no evidence for histotype specificity. Prexasertib as a single agent at 7.5 mg/kg consistently slowed tumor growth but failed to induce regressions in the 9 xenograft lines tested. Prexasertib at 10 mg/kg induced PR or CR in 6 of 6 neuroblastoma, 1 of 2 rhabdomyosarcoma, 0 of 1 Ewing sarcoma, and 0 of 2 osteosarcoma xenografts tested, and it induced a MCR in a rhabdoid tumor xenograft. Prexasertib single agent activity was comparable to that observed for single agent IR. The combination of IR and prexasertib (4 mg/kg) was significantly better than prexasertib (10 mg/kg) at prolonging EFS for only 1 of 6 NB, and the combination was significantly better than single agent IR for only 1 of 6 NB. For the remaining 7 xenograft lines, the combination was significantly superior to single agent IR for 3 lines (2 OS and 1 rhabdoid tumor), but the combination was significantly inferior to prexasertib (10 mg/kg) for the rhabdoid tumor line. Prexasertib had no effect as a single agent or in combination with XRT for two orthotopic medulloblastoma xenografts. Conclusions: Prexasertib at 10 mg/kg shows consistent tumor regressing activity as a single agent for neuroblastoma xenografts, confirming recently published data (Lowery, et al. CCR 23: 4354-63, 2017), and it shows tumor regressing activity for other histotypes. The limited activity of prexasertib at 7.5 mg/kg suggests a dose-response effect and that clinical trial design should seek the highest tolerated dose in children. For most xenografts, the combination of IR with prexasertib (at a reduced dose as required for tolerability) was no better than either single agent prexasertib at an optimized dose/schedule or single agent IR. An important area for future research is identifying agents that can be effectively combined with prexasertib for neuroblastoma and other childhood cancers. (Supported by NCI Grants/Contracts: CA199222, CA199221; CA199297; CA199288; CA199287; NO1-CM-42216) Citation Format: Kristina A. Cole, Peter J. Houghton, Raushan T. Kurmasheva, Richard Gorlick, E. Anders Kolb, Min Kang, C. Patrick Reynolds, Xiao-Nan Li, Holly Lindsay, Stephen W. Erickson, Yuelong Guo, Beverly A. Teicher, Malcolm A. Smith, John M. Maris. Pediatric Preclinical Testing Consortium evaluation of the CHK1 inhibitor prexasertib [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-B13.

The Roles of P53 and Its Family Proteins, P63 and P73, in the DNA Damage Stress Response in Organogenesis-Stage Mouse Embryos.

Members of the P53 transcription factor family, P53, P63, and P73, play important roles in normal development and in regulating the expression of genes that control apoptosis and cell cycle progression in response to genotoxic stress. P53 is involved in the DNA damage response pathway that is activated by hydroxyurea in organogenesis-stage murine embryos. The extent to which P63 and P73 contribute to this stress response is not known. To address this question, we examined the roles of P53, P63, and P73 in mediating the response of Trp53-positive and Trp53-deficient murine embryos to a single dose of hydroxyurea (400 mg/kg) on gestational day 9. Hydroxyurea treatment downregulated the expression of Trp63 and upregulated Trp73 in the absence of effects on the levels of Trp53 transcripts; Trp73 upregulation was P53-dependent. At the protein level, hydroxyurea treatment increased the levels and phosphorylation of P53 in the absence of effects on P63 and P73. Upregulation of the expression of genes that regulate cell cycle arrest and apoptosis, Cdkn1a, Rb1, Fas, Trp53inp1, and Pmaip1, was P53-dependent in hydroxyurea-treated embryos. The increase in cleaved caspase-3 and cleaved mammalian sterile-20-like-1 kinase levels induced by hydroxyurea was also P53-dependent; in contrast, the increase in phosphorylated H2AX, a marker of DNA double-strand breaks, in response to hydroxyurea treatment was only partially P53-dependent. Together, our data show that P53 is the principal P53 family member that is activated in the embryonic DNA damage response.

Adiponectin: A prosurvival and proproliferation signal that increases bovine mammary epithelial cell numbers and protects them from endoplasmic reticulum stress responses.

Cell-cell interactions between epithelial and stromal cells are predominant in the mammary gland, and various stromal cell-derived factors can elicit mitogenic responses in adjacent epithelial cells. Adiponectin is a hormone secreted mainly by adipocytes that mediates stromal-epithelial interactions in a number of tissues. Adiponectin receptors are expressed by bovine mammary epithelial cells, but the regulatory effects of adiponectin on the development and function of the mammary gland remain unclear. We therefore sought to investigate the effects of adiponectin on bovine mammary epithelial (MAC-T) cells and the regulatory mechanisms that underlie these adiponectin-induced actions. Our results revealed an increase in MAC-T cell proliferation and cell cycle progression in response to adiponectin. The expression of nuclear proliferating cell nuclear antigen (PCNA) and cyclin D1 was induced in MAC-T cells, and intracellular signaling molecules such as serine/threonine protein kinase (AKT), 70 kDa ribosomal S6 kinase (P70S6K), ribosomal protein S6 (S6), extracellular signal-regulated kinases 1 and 2 (ERK1/2), 90 kDa ribosomal S6 kinase (P90S6K), and cyclin D1 were activated in a dose-dependent manner. The abundance of adiponectin-induced signaling proteins was suppressed following inhibition of AKT or ERK1/2 mitogen-activated protein kinase (MAPK) signaling. In addition, inhibition of AKT or ERK1/2 signaling significantly reduced adiponectin-stimulated MAC-T cell proliferation. Furthermore, adiponectin reduced tunicamycin-induced expression and activation of endoplasmic reticulum stress-related proteins in MAC-T cells and attenuated the repressive effect of tunicamycin on proliferation of MAC-T cells. Collectively, these results suggest that adiponectin-mediated signaling may affect the development and function of the mammary gland in dairy cows by increasing mammary epithelial cell numbers. These findings may result in important implications for improving our fundamental understanding of lactation physiology in livestock species.

Checkpoint kinase 1 is essential for normal B cell development and lymphomagenesis

Checkpoint kinase 1 (CHK1) is critical for intrinsic cell cycle control and coordination of cell cycle progression in response to DNA damage. Despite its essential function, CHK1 has been identified as a target to kill cancer cells and studies using Chk1 haploinsufficient mice initially suggested a role as tumor suppressor. Here, we report on the key role of CHK1 in normal B-cell development, lymphomagenesis and cell survival. Chemical CHK1 inhibition induces BCL2-regulated apoptosis in primary as well as malignant B-cells and CHK1 expression levels control the timing of lymphomagenesis in mice. Moreover, total ablation of Chk1 in B-cells arrests their development at the pro-B cell stage, a block that, surprisingly, cannot be overcome by inhibition of mitochondrial apoptosis, as cell cycle arrest is initiated as an alternative fate to limit the spread of damaged DNA. Our findings define CHK1 as essential in B-cell development and potent target to treat blood cancer.

Abstract 1: Sprr2b Drives Proliferation of Cardiac Fibroblasts by Relieving p53-mediated Cell Cycle Inhibition

Pathological cardiac remodeling is initially a compensatory attempt to increase cardiac output, but ultimately leads to the development of fibrosis, a form of scarring that contributes to heart failure (HF). In contrast, physiological cardiac remodeling in response to exercise is not associated with the development of fibrosis and typically remains compensatory. Understanding how cardiac fibroblasts (CF), the primary source of extracellular matrix in the heart, respond to pathological and physiological cues might lead to novel approaches to limit the maladaptive effects of pathological cardiac remodeling. We performed RNA sequencing to define genes that are differentially regulated in CF during physiological (swimming) or pathological (pressure overload) remodeling. This study revealed that cardiac expression of the s mall pr oline r ich 2b ( Sprr2b) gene is restricted to CFs and is significantly elevated in disease and lost in exercise. We demonstrate that SPRR2B drives CF proliferation, but not myofibroblast differentiation, in response to pathological cues. SPRR2B facilitates an interaction between MDM2 and USP7, a nuclear deubiquitinase that leads to proteasomal degradation of p53. SPRR2B-USP7-MDM2 complex formation and p53 degradation is at least partially dependent upon phosphorylation of SPRR2B by Src-family NRTKs. SPRR2B thus relieves p53-mediated constraints on cell cycle progression in response to Src-dependent signaling, leading to CF accumulation. Importantly, SPRR2B expression is elevated in cardiac tissue from human HF patients relative to individuals without heart disease and positively correlates with a proliferative, activated gene expression profile in HF patient CF. Treatment of human HF fibroblasts with IGF-1/H 2 O 2 to mimic physiological cues significantly abrogated SPRR2B expression and increased expression of p53-dependent cell cycle checkpoint genes, which correlated with a less activated phenotype. Taken together, this study defines a unique tissue-specific role of Sprr2b in driving pathological CF cell cycle progression that may underlie the development of cardiac fibrosis.

In vitro와 ex vivo 혈관신생 모델에서 황련 냉수추출물의 신생혈관 억제효과

혈관신생, 즉 새로운 혈관형성은 종양의 성장과 전이에 중요한 역할을 한다고 알려져 있으며, 암 치료에 중요한 목표물이 되고 있다. 이 연구의 목적은 황련 냉수추출물의 혈관신생 억제작용의 효과를 밝히고 항암제로서의 가능성을 평가하고자 한다. Ex vivo rat aortic ring assay 실험결과 신생혈관성장을 억제하는 결과를 확인하였고, 이것을 통해 황련 냉수추출물이 혈관신생과정의 주요한 단계와 내피세포의 증식, 이동, 침투, 혈관내피세포자극인자에 의한 반응으로 모세관 모양의 관 형성작용을 억제함을 알아내었다. 또한 황련 냉수추출물을 처리하였을 때, 세포주기가 억제되고 VEGF에 의한 반응으로 인해 G0/G1 주기에서 S 주기로 가는 과정을 예방하고, VEGF에 의해 활성화가 유도되는 MMP-2, MMP-9이 감소되었다. 따라서 이들의 결과는 황련 냉수추출물이 종양의 발달 단계 중 혈관신생을 방해하는 잠재적인 항암약물의 소재로 고려될 수 있음을 제안한다. Angiogenesis, the formation of new blood vessels, plays an important role in tumor growth and metastasis; therefore, it has become an important target in cancer therapy. Novel anticancer pharmaceutical products that have relatively few side effects or are non-cytotoxic must be developed, and such products may be obtained from traditional herbal medicines. Coptis chinensis Franch. is an herb used in traditional medicine for the treatment of inflammatory diseases and diabetes. However, potential antiangiogenic effects of C. chinensis water extract (CCFWE) have not yet been studied. The purpose of this study was to determine the antiangiogenic effect of CCFWE in order to evaluate its potential for an anticancer drug. We found that the treatment with CCFWE inhibited the major steps of the angiogenesis process, such as the endothelial cell proliferation, migration, invasion, and capillary-like tube formation in response to vascular endothelial growth factor (VEGF), and also resulted in the growth inhibition of new blood vessels in an ex vivo rat aortic ring assay. We also observed that CCFWE treatment arrested the cell cycle at the G0/G1 phase, preventing the G0/G1 to S phase cell cycle progression in response to VEGF. In addition, the treatment reduced the VEGF-induced activation of matrix metalloproteinases 2 and 9. Taken together, these findings indicate that CCFWE should be considered a potential anticancer therapy against pathological conditions where angiogenesis is stimulated during tumor development.