Retinoblastoma Protein Family Research Articles

Abstract GMM-053: ROLE OF LATS AND DYRK1A KINASES IN THE PATHOGENESIS AND TREATMENT OF HGSOC

Abstract Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy, with 75% of women presenting with metastatic disease at the time of diagnosis. High Grade Serous Ovarian Carcinoma (HGSOC) is the most common and aggressive type of epithelial ovarian cancer, with 5-year survival rate of 30% for most patients. Developing of accurate experimental models of HGSOC is necessary to elucidate the disease pathogenesis and to evaluate new treatments. Our study is aimed at determining the roles of the Large Tumor Suppressor 1 and 2 kinases (LATS1/2), and their downstream effector Dual-specificity tyrosine(Y)-Regulated Protein Kinase 1A (DYRK1A), in HGSOC pathogenesis and treatment sensitivity. The Cancer Genome Atlas study revealed copy number losses of the LATS1, LATS2 and DYRK1A genes in 65%, 59% and 38% of HGSOC cases, respectively. Previous studies found that LATS1/2 and DYRK1A mediate a crosstalk between the Hippo tumor suppressor pathway and the G0/G1 cell cycle checkpoint. However, the role of these kinases in HGSOC pathogenesis is not known. We found that loss of both LATS1 and LATS2, or DYRK1A results in increased proliferation of SKOV3 ovarian cancer cells in 2D or 3D cultures, mediated in part by disruption of the DREAM complex that represses cell cycle-regulated genes. Interestingly, we observed upregulation of cyclin D1 and increased phosphorylation of retinoblastoma (RB) family proteins in SKOV3 cells depleted of LATS1/2 or DYRK1A kinases, indicative of aberrant activation of CDK4/6. Similar changes were observed upon depletion of LATS1/2 or DYRK1A using non-transformed human fallopian epithelial cells, suggesting that genetic losses of these kinases could contribute to the HGSOC pathogenesis. Furthermore, we tested whether an aberrant activation of CDK4/6 in SKOV3 cells with low levels of LATS1/2 or DYRK1A could increase their sensitivity to pharmacological CDK4/6 inhibitors such as palbociclib. Using metabolic cell proliferation assays, we found that loss of LATS1/2 or DYRK1A resulted in a significant decrease of palbociclib IC50 in SKOV3 cells. Interestingly, palbociclib treatment increased the DREAM assembly in these cells, resulting in downregulation of DREAM-regulated cell cycle factors such as cyclin A or B-Myb. Our results show that loss of LATS1/2 or DYRK1A could contribute to HGSOC pathogenesis by CDK4/6-mediated disruption of RB family function. Further studies are underway to validate our findings in vivo using orthotopic ovarian tumor xenografts in mice and a bioluminescence imaging approach. In addition, these models will be used for validation of novel therapeutic approaches for HGSOC, such as palbociclib, as well as for the mechanistic studies of the EOC pathogenesis. Citation Format: Fatmata Sesay, Siddharth Saini, Larisa Litovchick. ROLE OF LATS AND DYRK1A KINASES IN THE PATHOGENESIS AND TREATMENT OF HGSOC [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-053.

PS927 PHOSPHOCHROMATOME ANALYSIS UNCOVERS UNEXPECTED ROLES OF CDK6 IN REGULATING TRANSCRIPTION

Background:Cyclin‐dependent kinase 6 (CDK6) together with its close homologue CDK4 enables the progression of cells through the early G1 phase of the cell cycle. CDK4/6‐cyclin D complexes phosphorylate members of the retinoblastoma (Rb) protein family, allowing for the release of E2F transcription factors that induce genes required for cell cycle progression and DNA replication. Alterations of the CDK4/6‐Rb pathway are frequently observed in human malignancies. Recent studies focusing on acute lymphoid leukemia (ALL) and acute myeloid leukemia (AML) have uncovered a novel role for CDK6 in regulating transcription. Our group has identified CDK6 as part of chromatin‐bound complexes which drive transcription of disease–relevant genes including the cell cycle inhibitor p16INK4a, the angiogenesis‐promoting factor VEGF‐A (vascular endothelial factor A) and the proto‐oncogene FLT3 (Fms like tyrosine kinase 3).Aims:The aim of this study is to identify novel binding partners of CDK6 on chromatin and to investigate their target gene sets. A better understanding of CDK6 functionality in tumor formation and promotion may help in the design and selection of adequate treatment options.Methods:For further investigation of CDK6́s role in transcription, we transduced murine Cdk6 +/+ and Cdk6 −/− primary bone marrow with the oncogene Bcr/Abl p185. To systematically gain insights on how CDK6 takes influence on chromatin‐binding and activity of transcription factors we performed RNA‐seq paralleled by mass spectrometry‐based phosphoproteomics of chromatin‐bound proteins (Phospho‐Chromatome).Results:RNA‐seq analysis of Cdk6 +/+ and Cdk6 −/− BCR‐ABL transformed cells identified more than 1700 genes that are differentially expressed. Phospho‐Chromatome studies performed in parallel revealed significant changes in the phosphorylation status of the transcription factors ZBTB7A, EBF1 and YY1. In vitro kinase assays verified a direct phosphorylation of ZBTB7A and EBF1 by active CDK6/CyclinD3 complexes.Summary/Conclusion:We show that CDK6 is broadly associated with chromatin where it fulfils different tasks: beside its well‐known role in the E2F‐release it enhances the phosphorylation of various transcription factors and chromatin‐regulating enzymes. This results in pronounced changes in associated gene expression. Therefor our data indicate that the function of CDK6 is not limited to cell cycle progression but regulates a plethora of tumor‐relevant transcriptional programs. Future therapies may consider this unexpected role and even exploit it to improve personalized and cancer‐specific treatment protocols.

PTPN14 degradation by high-risk human papillomavirus E7 limits keratinocyte differentiation and contributes to HPV-mediated oncogenesis

High-risk human papillomavirus (HPV) E7 proteins enable oncogenic transformation of HPV-infected cells by inactivating host cellular proteins. High-risk but not low-risk HPV E7 target PTPN14 for proteolytic degradation, suggesting that PTPN14 degradation may be related to their oncogenic activity. HPV infects human keratinocytes but the role of PTPN14 in keratinocytes and the consequences of PTPN14 degradation are unknown. Using an HPV16 E7 variant that can inactivate retinoblastoma tumor suppressor (RB1) but cannot degrade PTPN14, we found that high-risk HPV E7-mediated PTPN14 degradation impairs keratinocyte differentiation. Deletion of PTPN14 from primary human keratinocytes decreased keratinocyte differentiation gene expression. Related to oncogenic transformation, both HPV16 E7-mediated PTPN14 degradation and PTPN14 deletion promoted keratinocyte survival following detachment from a substrate. PTPN14 degradation contributed to high-risk HPV E6/E7-mediated immortalization of primary keratinocytes and HPV+ but not HPV- cancers exhibit a gene-expression signature consistent with PTPN14 inactivation. We find that PTPN14 degradation impairs keratinocyte differentiation and propose that this contributes to high-risk HPV E7-mediated oncogenic activity independent of RB1 inactivation.

Mitochondrial protein E2F3d, a distinctive E2F3 product, mediates hypoxia-induced mitophagy in cancer cells

Mitochondrial damage is caused by changes in the micro-environmental conditions during tumor progression. Cancer cells require mechanisms for mitochondrial quality control during this process; however, how mitochondrial integrity is maintained is unclear. Here we show that E2F3d, a previously unidentified E2F3 isoform, mediates hypoxia-induced mitophagy in cancer cells. Aberrant activity and expression of the E2F3 transcription factor is frequently observed in many cancer cells. Loss of retinoblastoma (Rb) protein family function increases the expression of E2F3d and E2F3a. E2F3d localizes to the outer mitochondrial membrane and its cytosolic domain contains an LC3-interacting region motif. Overexpression of E2F3d induces mitochondrial fragmentation and mitophagy, suggesting that E2F3d plays an important role in mitophagy. Furthermore, depletion of E2F3s attenuates hypoxia-induced mitophagy and increases intracellular levels of reactive oxygen species, which is reversed by the reintroduction of E2F3d. This study presents another key player that regulates mitochondrial quality control in cancer cells.

Heat shock protein 27 promotes cell cycle progression by down-regulating E2F transcription factor 4 and retinoblastoma family protein p130

Heat shock protein 27 (HSP27) protects cells under stress. Here, we demonstrate that HSP27 also promotes cell cycle progression of MRC-5 human lung fibroblast cells. Serum starvation for 24 h induced G1 arrest in these cells, and upon serum refeeding, the cells initiated cell cycle progression accompanied by an increase in HSP27 protein levels. HSP27 levels peaked at 12 h, and transcriptional up-regulation of six G2/M-related genes (CCNA2, CCNB1, CCNB2, CDC25C, CDCA3, and CDK1) peaked at 24-48 h. siRNA-mediated HSP27 silencing in proliferating MRC-5 cells induced G2 arrest coinciding with down-regulation of these six genes. Of note, the promoters of all of these genes have the cell cycle-dependent element and/or the cell cycle gene-homology region. These promoter regions are known to be bound by the E2F family proteins (E2F-1 to E2F-8) and retinoblastoma (RB) family proteins (RB1, p107, and p130), among which E2F-4 and p130 were strongly up-regulated in HSP27-knockdown cells. E2F-4 or p130 knockdown concomitant with the HSP27 knockdown rescued MRC-5 cells from G2 arrest and up-regulated the six cell cycle genes. Moreover, we observed cellular senescence in MRC-5 cells on day 3 after the HSP27 knockdown, as evidenced by increased senescence-associated β-gal activity and up-regulated inflammatory cytokines. The cellular senescence was also suppressed by the concomitant knockdown of E2F-4/HSP27 or p130/HSP27. Our findings indicate that HSP27 promotes cell cycle progression of MRC-5 cells by suppressing expression of the transcriptional repressors E2F-4 and p130.

Abstract B49: The role of LATS kinases in regulation of CDK4/6 in ovarian cancer

Abstract High-grade ovarian carcinoma (HGSOC) is the most lethal gynecologic cancer with little improvement in survival over the last decade. Identification of the “druggable” cancer-driving pathways could facilitate the development of effective treatments for HGSOC patients. Oncogenomic analyses of HGSOC tumors revealed widespread genome alterations but only a few recurrent mutations that could be targeted by either approved or experimental therapeutics. We observed copy number losses of genes encoding LATS1 and LATS2 tumor suppressor protein kinases in approximately 60% of HGSOC tumors analyzed by TCGA study. Loss of LATS kinases could contribute to cancer pathogenesis because of their important role in the Hippo tumor suppressor pathway that is known to control cell invasion, migration, and proliferation though inhibition of the oncogenic transcription factors YAP and TAZ. Here, we describe an alternative mechanism by which LATS kinases can control cell proliferation and discuss the implications of this finding for HGSOC treatment. In previous studies, LATS2 kinase was shown to promote growth suppressor function of retinoblastoma family. LATS2 phosphorylation of DYRK1A protein kinase promoted assembly of the transcriptional repressor DREAM. DREAM assembles during G0/G1 when RB-like p130 binds E2F4, DP1 and a stable complex of five proteins, including RBBP4, LIN9, LIN37, LIN52, and LIN54 (MuvB core), and inhibits the expression of more than 800 cell cycle-regulated genes. Here, we report that LATS1 and LATS2 function redundantly in the regulation of DREAM in SKOV3 ovarian cancer cells and in the nontransformed fallopian tube epithelial (FTE) cells. Downregulation of LATS1 and LATS2 expression resulted in increased proliferation and promoted transformed phenotype of these cells, suggesting that loss of these genes could contribute to ovarian cancer aggressiveness. Interestingly, we observed that depletion of both LATS kinases in these cells resulted in markedly increased phosphorylation of the retinoblastoma family proteins pRB and p130. This phosphorylation was likely mediated by CDK4/6 kinases because it was completely abolished by treatment of cells with the highly selective CDK4/6 inhibitor, PD-0332991 (palbociclib). We investigated the mechanism of CDK4/6 activation, and found that downregulation of LATS kinases in SKOV3 or FTE cells resulted in a markedly increased expression of cyclin D1 at the protein, but not at the mRNA level. In a recent study, loss of the CDK4/6 inhibitor, p16, correlated with increased sensitivity of ovarian cancer cell lines to palbociclib. Therefore, we tested whether aberrant activation of CDK4/6 in LATS1/2-depleted cells could similarly predispose ovarian cancer cells to palbociclib growth inhibition. Indeed, downregulation of LATS kinases in SKOV3 cells resulted in 3-fold decrease of palbociclib IC50 compared to control cell lines. Importantly, clinical trials using palbociclib and analogous drugs in several types of cancer revealed a wide range of outcomes, emphasizing significance of research that will help to predict tumor sensitivity. Currently, there are no HGSOC-specific clinical trials using palbociclib. Our findings reported here support the concept that frequent genetic losses of LATS1 and LATS2 kinases in HGSOC could provide a rationale for using CDK4/6 inhibitors, such as palbociclib, for treatment of patients suffering from this disease. Citation Format: Siddharth Saini, Fatmata Sesay, Larisa Litovchick. The role of LATS kinases in regulation of CDK4/6 in ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B49.

RBL2/p130 is a direct AKT target and is required to induce apoptosis upon AKT inhibition in lung cancer and mesothelioma cell lines.

The retinoblastoma (RB) protein family includes RB1/p105, RBL1/p107, and RBL2/p130, which are key factors in cell-cycle regulation and stand at the crossroads of multiple pathways dictating cell fate decisions. The role of RB proteins in apoptosis is controversial because they can inhibit or promote apoptosis depending on the context, on the apoptotic stimuli and on their intrinsic status, impacting on the response to antitumoral treatments. Here we identified RBL2/p130 as a direct substrate of the AKT kinase, a key antiapoptotic factor hyperactive in multiple cancer types. We showed that RBL2/p130 and AKT1 physically interact and AKT phosphorylates RBL2/p130 Ser941, located in the pocket domain, but not when this residue is mutated into Ala. We found that pharmacological inhibition of AKT, through the highly selective AKT inhibitor VIII (AKTiVIII), impairs RBL2/p130 Ser941 phosphorylation and increases RBL2/p130 stability, mRNA expression and nuclear levels in both lung cancer and mesothelioma cell lines, mirroring the more extensively studied effects on the p27 cell-cycle inhibitor. Consistently, AKT inhibition reduced cell viability, induced cell accumulation in G0/G1, and triggered apoptosis, which proved to be largely dependent on RBL2/p130 itself, as shown upon RBL2/p130 silencing. AKT inhibition induced RBL2/p130-dependent apoptosis also in HEK-293 cells, in which re-expression of a short hairpin-resistant RBL2/p130 was able to rescue AKTiVIII-induced apoptosis upon RBL2/p130 silencing. Our data also showed that the combination of AKT and cyclin-dependent kinases (CDK) inhibitors, which converge on the re-activation of RBL2/p130 antitumoral potential, could be a promising anticancer strategy.

P27Kip1 regulates alpha-synuclein expression

Alpha-synuclein (α-SYN) is the main component of anomalous protein aggregates (Lewy bodies) that play a crucial role in several neurodegenerative diseases (synucleinopathies) like Parkinson’s disease and multiple system atrophy. However, the mechanisms involved in its transcriptional regulation are poorly understood. We investigated here the role of the cyclin-dependent kinase (Cdk) inhibitor and transcriptional regulator p27Kip1 (p27) in the regulation of α-SYN expression. We observed that selective deletion of p27 by CRISPR/Cas9 technology in neural cells resulted in increased levels of α-SYN. Knock-down of the member of the same family p21Cip1 (p21) also led to increased α-SYN levels, indicating that p27 and p21 collaborate in the repression of α-SYN transcription. We demonstrated that this repression is mediated by the transcription factor E2F4 and the member of the retinoblastoma protein family p130 and that it is dependent of Cdk activity. Chromatin immunoprecipitation analysis revealed specific binding sites for p27, p21 and E2F4 in the proximal α-SYN gene promoter. Finally, luciferase assays revealed a direct action of p27, p21 and E2F4 in α-SYN gene expression. Our findings reveal for the first time a negative regulatory mechanism of α-SYN expression, suggesting a putative role for cell cycle regulators in the etiology of synucleinopathies.

PARP1 facilitates EP300 recruitment to the promoters of the subset of RBL2-dependent genes

Differentiation of human monocytes is associated with proliferation arrest resulting from activation of the inter alia retinoblastoma protein family of gene repressors, which target gene promoters in an E2F-dependent manner. To investigate RBL2 contribution to defining monocyte phenotype and function, we used primer libraries. We identified genes encoding two surface receptors (CXCR1 and IL17RE) and two TLR signaling mediators (CD86 and NFKB2) that are repressed by the RBL2-E2F4-HDAC1-BRM complex. Surprisingly, PARP1 co-regulated 24 out of the 28 identified genes controlled by RBL2. Upon RBL2 silencing, PARP1 was recruited to one subset of RBL2-dependent genes, represented by MAP2K6 and MAPK3. RBL2 silencing also restored PARP1 transcription. Gene promoters enriched in PARP1 were characterized by increased histone acetylation and the replacement of HDAC1 with EP300. While PARP1 was dispensable for HDAC1 dissociation, EP300 was found only at gene promoters enriched in PARP1. EP300 activated transcription of PARP1/RBL2 co-regulated genes, but not genes solely controlled by RBL2. DNA was a prerequisite to the formation of an immunoprecipitated PARP1-EP300 complex, suggesting that PARP1 enabled EP300 binding, which in turn activated gene transcription. Notably, PARP1 overexpression failed to overcome the inhibitory effect of RBL2 on MAP2K6 and MAPK3 transcription. The same interdependence was observed in proliferating cancer cells; the low abundance of RBL2 resulted in PARP1-mediated EP300 recruitment to promoters of the MAP2K6 and MAPK3 genes. We conclude that RBL2 may indirectly regulate transcription of some genes by controlling PARP1-mediated EP300 recruitment.

Dnmt1-dependent Chk1 pathway suppression is protective against neuron division.

Neuronal differentiation and cell-cycle exit are tightly coordinated, even in pathological situations. When pathological neurons re-enter the cell cycle and progress through the S phase, they undergo cell death instead of division. However, the mechanisms underlying mitotic resistance are mostly unknown. Here, we have found that acute inactivation of retinoblastoma (Rb) family proteins (Rb, p107 and p130) in mouse postmitotic neurons leads to cell death after S-phase progression. Checkpoint kinase 1 (Chk1) pathway activation during the S phase prevented the cell death, and allowed the division of cortical neurons that had undergone acute Rb family inactivation, oxygen-glucose deprivation (OGD) or in vivo hypoxia-ischemia. During neurogenesis, cortical neurons became protected from S-phase Chk1 pathway activation by the DNA methyltransferase Dnmt1, and underwent cell death after S-phase progression. Our results indicate that Chk1 pathway activation overrides mitotic safeguards and uncouples neuronal differentiation from mitotic resistance.

CDK4 protein is degraded by anaphase-promoting complex/cyclosome in mitosis and reaccumulates in early G1 phase to initiate a new cell cycle in HeLa cells

CDK4 regulates G1/S phase transition in the mammalian cell cycle by phosphorylating retinoblastoma family proteins. However, the mechanism underlying the regulation of CDK4 activity is not fully understood. Here, we show that CDK4 protein is degraded by anaphase-promoting complex/cyclosome (APC/C) during metaphase-anaphase transition in HeLa cells, whereas its main regulator, cyclin D1, remains intact but is sequestered in cytoplasm. CDK4 protein reaccumulates in the following G1 phase and shuttles between the nucleus and the cytoplasm to facilitate the nuclear import of cyclin D1. Without CDK4, cyclin D1 cannot enter the nucleus. Point mutations that disrupt CDK4 and cyclin D1 interaction impair the nuclear import of cyclin D1 and the activity of CDK4. RNAi knockdown of CDK4 also induces cytoplasmic retention of cyclin D1 and G0/G1 phase arrest of the cells. Collectively, our data demonstrate that CDK4 protein is degraded in late mitosis and reaccumulates in the following G1 phase to facilitate the nuclear import of cyclin D1 for activation of CKD4 to initiate a new cell cycle in HeLa cells.

Conservation and divergence of C-terminal domain structure in the retinoblastoma protein family

The retinoblastoma protein (Rb) and the homologous pocket proteins p107 and p130 negatively regulate cell proliferation by binding and inhibiting members of the E2F transcription factor family. The structural features that distinguish Rb from other pocket proteins have been unclear but are critical for understanding their functional diversity and determining why Rb has unique tumor suppressor activities. We describe here important differences in how the Rb and p107 C-terminal domains (CTDs) associate with the coiled-coil and marked-box domains (CMs) of E2Fs. We find that although CTD-CM binding is conserved across protein families, Rb and p107 CTDs show clear preferences for different E2Fs. A crystal structure of the p107 CTD bound to E2F5 and its dimer partner DP1 reveals the molecular basis for pocket protein-E2F binding specificity and how cyclin-dependent kinases differentially regulate pocket proteins through CTD phosphorylation. Our structural and biochemical data together with phylogenetic analyses of Rb and E2F proteins support the conclusion that Rb evolved specific structural motifs that confer its unique capacity to bind with high affinity those E2Fs that are the most potent activators of the cell cycle.

Genomic profiles of low-grade murine gliomas evolve during progression to glioblastoma.

Gliomas are diverse neoplasms with multiple molecular subtypes. How tumor-initiating mutations relate to molecular subtypes as these tumors evolve during malignant progression remains unclear. We used genetically engineered mouse models, histopathology, genetic lineage tracing, expression profiling, and copy number analyses to examine how genomic tumor diversity evolves during the course of malignant progression from low- to high-grade disease. Knockout of all 3 retinoblastoma (Rb) family proteins was required to initiate low-grade tumors in adult mouse astrocytes. Mutations activating mitogen-activated protein kinase signaling, specifically KrasG12D, potentiated Rb-mediated tumorigenesis. Low-grade tumors showed mutant Kras-specific transcriptome profiles but lacked copy number mutations. These tumors stochastically progressed to high-grade, in part through acquisition of copy number mutations. High-grade tumor transcriptomes were heterogeneous and consisted of 3 subtypes that mimicked human mesenchymal, proneural, and neural glioblastomas. Subtypes were confirmed in validation sets of high-grade mouse tumors initiated by different driver mutations as well as human patient-derived xenograft models and glioblastoma tumors. These results suggest that oncogenic driver mutations influence the genomic profiles of low-grade tumors and that these, as well as progression-acquired mutations, contribute strongly to the genomic heterogeneity across high-grade tumors.

Rbf Regulates Drosophila Spermatogenesis via Control of Somatic Stem and Progenitor Cell Fate in the Larval Testis.

SummaryThe Drosophila testis has been fundamental to understanding how stem cells interact with their endogenous microenvironment, or niche, to control organ growth in vivo. Here, we report the identification of two independent alleles for the highly conserved tumor suppressor gene, Retinoblastoma-family protein (Rbf), in a screen for testis phenotypes in X chromosome third-instar lethal alleles. Rbf mutant alleles exhibit overproliferation of spermatogonial cells, which is phenocopied by the molecularly characterized Rbf11 null allele. We demonstrate that Rbf promotes cell-cycle exit and differentiation of the somatic and germline stem cells of the testes. Intriguingly, depletion of Rbf specifically in the germline does not disrupt stem cell differentiation, rather Rbf loss of function in the somatic lineage drives overproliferation and differentiation defects in both lineages. Together our observations suggest that Rbf in the somatic lineage controls germline stem cell renewal and differentiation non-autonomously via essential roles in the microenvironment of the germline lineage.

Deregulation of the pRb-E2F4 axis alters epidermal homeostasis and favors tumor development.

E2F/RB activity is altered in most human tumors. The retinoblastoma family of proteins plays a key role in regulating the progression of the cell cycle from the G1 to S phases. This is achieved through negative regulation of E2F transcription factors, important positive regulators of cell cycle entry. E2F family members are divided into two groups: activators (E2F1-E2F3a) and repressors (E2F3b-E2F8). E2F4 accounts for a large part of the E2F activity and is a main E2F repressor member in vivo. Perturbations in the balance from quiescence towards proliferation contribute to increased mitotic gene expression levels frequently observed in cancer. We have previously reported that combined Rb1-Rbl1 or Rb1-E2f1 ablation in epidermis produces important alterations in epidermal proliferation and differentiation, leading to tumor development. However, the possible roles of E2F4 in this context are still to be determined. Here, we show the absence of any discernible phenotype in the skin of mice lacking of E2f4. In contrast, the inducible loss of Rb1 in the epidermis of E2F4-null mice produced multiple skin abnormalities including altered differentiation and proliferation, spontaneous wounds, carcinoma in situ development and stem cell perturbations. All these phenotypic alterations are associated with extensive gene expression changes, the induction of c-myc and the Akt activation. Moreover the whole transcriptome analyses in comparison with previous models generated also revealed extensive changes in multiple repressive complexes and in transcription factor activity. These results point to E2F4 as a master regulator in multiple steps of epidermal homeostasis in Rb1 absence.

High-Risk Human Papillomavirus E7 Proteins Target PTPN14 for Degradation.

ABSTRACTThe major transformation activity of the high-risk human papillomaviruses (HPV) is associated with the E7 oncoprotein. The interaction of HPV E7 with retinoblastoma family proteins is important for several E7 activities; however, this interaction does not fully account for the high-risk E7-specific cellular immortalization and transformation activities. We have determined that the cellular non-receptor protein tyrosine phosphatase PTPN14 interacts with HPV E7 from many genus alpha and beta HPV types. We find that high-risk genus alpha HPV E7, but not low-risk genus alpha or beta HPV E7, is necessary and sufficient to reduce the steady-state level of PTPN14 in cells. High-risk E7 proteins target PTPN14 for proteasome-mediated degradation, which requires the ubiquitin ligase UBR4, and PTPN14 is degraded by the proteasome in HPV-positive cervical cancer cell lines. Residues in the C terminus of E7 interact with the C-terminal phosphatase domain of PTPN14, and interference with the E7-PTPN14 interaction restores PTPN14 levels in cells. Finally, PTPN14 degradation correlates with the retinoblastoma-independent transforming activity of high-risk HPV E7.

Cell division of neurons in an oxygen-glucose deprivation model

Event Abstract Back to Event Cell division of neurons in an oxygen-glucose deprivation model Itsuki Ajioka1, 2* and Mio Oshikawa1 1 Tokyo Medical and Dental University (TMDU), Center for Brain Integration Research (CBIR), Japan 2 JST, PRESTO, Japan Although neurons are able to re-enter the cell cycle in pathological situations such as neurodegeneration and stroke, they are tightly protected from cell division and undergo cell death after S phase progression. Neurons become permanently post-mitotic immediately after cell cycle exit during development, however, the mechanism underlying non-dividing feature is mostly unknown. The Retinoblastoma protein family (Rb, p107, p130) plays a central role in preventing cells from entering the S phase. When Rb family expression is lost in neuronal progenitor cells, the subsequent coordination of cell-cycle exit and neuronal differentiation is lost, and neurons can divide in some cases such as retinoblastoma (Ajioka et al., 2007). We recently developed a technique for conditionally inactivating all of the Rb family members in mouse cortical progenitors, either before or immediately after cell-cycle exit, by electroporation with Cre-expressing plasmids containing a ubiquitous pCAG promoter or a neuron-specific pMAP2 promoter (Oshikawa et al., 2013). When the Rb family is inactivated using pCAG-Cre, immature neurons generated from the pCAG-induced Rb-TKO (Rb-/-; p107-/-; p130-/-) progenitors divide. In contrast, the pMAP2-induced Rb-TKO immature neurons enter the S phase, but undergo cell death. Thus, once progenitor daughter cells exit the cell-cycle and initiate neuronal differentiation, they are prevented from undergoing cell division, and maintain mitotic resistance even after acute Rb family inactivation. These findings led us to hypothesize that pathological neurons in the S phase undergo cell death by activating mitotic safeguards. To investigate whether such mitotic safeguard protects pathological neurons in the S phase from undergoing cell division, we used oxygen-glucose deprivation (OGD) model in which post-mitotic cortical neurons undergo cell death after S phase progression and Rb phosphorylation. We demonstrate the cell division of dying OGD neurons in the S phase by inactivating mitotic safeguards. Keywords: Cell Cycle, Cell Division, Phosphorylation, Neuron, retinoblastoma protein family Conference: 14th Meeting of the Asian-Pacific Society for Neurochemistry, Kuala Lumpur, Malaysia, 27 Aug - 30 Aug, 2016. Presentation Type: Poster Presentation Session Topic: 14th Meeting of the Asian-Pacific Society for Neurochemistry Citation: Ajioka I and Oshikawa M (2016). Cell division of neurons in an oxygen-glucose deprivation model. Conference Abstract: 14th Meeting of the Asian-Pacific Society for Neurochemistry. doi: 10.3389/conf.fncel.2016.36.00182 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 04 Aug 2016; Published Online: 11 Aug 2016. * Correspondence: Prof. Itsuki Ajioka, Tokyo Medical and Dental University (TMDU), Center for Brain Integration Research (CBIR), Tokyo, Tokyo, Japan, iajioka.cbir@tmd.ac.jp Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Itsuki Ajioka Mio Oshikawa Google Itsuki Ajioka Mio Oshikawa Google Scholar Itsuki Ajioka Mio Oshikawa PubMed Itsuki Ajioka Mio Oshikawa Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Abstract 1965: Hippo pathway restrains cell proliferation via assembly of the DREAM repressor complex

Abstract Hippo pathway mediates tumor suppression in various organisms by the mechanisms that are not fully understood. The canonical Hippo pathway converges on the pro-oncogenic transcription factors YAP and TAZ that are phosphorylated by LATS1 or LATS2 protein kinases, resulting in their proteasomal degradation. Hippo pathway is regulated by various extracellular signals including lysophosphatidic acid (LPA), a bioactive lipid that has a tumor-promoting activity. High levels of LPA in the malignant effusions correlate with increased epithelial ovarian cancer (EOC) aggressiveness. However, the mitogenic effect of LPA is not well understood. We hypothesized that the Hippo signaling can regulate cell proliferation though a novel mechanism involving retinoblastoma (RB) family proteins. Previous studies show that LATS2 contributes to the function of the DREAM (DP, RB-like, E2F, and MuvB core) complex that promotes the G0/G1 arrest by repressing more than 800 cell cycle-regulated genes. The DREAM complex formation depends on phosphorylation of LIN52 subunit of the MuvB core by DYRK1A protein kinase. DYRK1A activity towards LIN52 is promoted by LATS2 phosphorylation in vitro. However, it remains to be established whether the Hippo signaling regulates the function of DREAM complex in vivo. To understand the mechanism by which inhibition of the Hippo signaling can promote cell proliferation, we determined the effect of LPA on the DREAM complex in the ovarian cancer and the precursor cell lines. Using immunoprecipitation-Western blot assays, we show that the LPA treatment of serum-starving cells prevents the recruitment of RB-like p130 into the DREAM complex. Consequently, the cells escape from G0/G1 arrest as demonstrated by FACS analysis of the DNA synthesis. Since MuvB core also contributes to the expression of mitotic genes through binding to BMYB transcription factor, we show that this interaction is not disrupted by LPA. In support of the model that LPA effects are mediated by inhibition of the Hippo signaling, the RNAi knockdown of both LATS1 and LATS2 in ovarian cell lines also resulted in the impaired DREAM assembly. To characterize the mechanism by which LPA can interfere with the DREAM complex formation, we investigated the changes in LIN52 phosphorylation upon LPA treatment or LATS depletion. Our data support the model in which the disruption of the Hippo signaling inhibits the phosphorylation of LIN52, thereby preventing the formation of the DREAM complex and entry into G0/G1 arrest. Our findings highlight a novel mechanism by which inhibition of the Hippo tumor suppressor pathway can promote cancer cell proliferation. Citation Format: Larisa Litovchick, Siddharth Saini. Hippo pathway restrains cell proliferation via assembly of the DREAM repressor complex. [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 1965. doi:10.1158/1538-7445.AM2015-1965

Retinoblastoma family proteins: New players in DNA repair by non-homologous end-joining

ABSTRACTLoss of retinoblastoma protein (RB1) function is a major driver in cancer development. We have recently reported that, in addition to its well-documented functions in cell cycle and fate control, RB1 and its paralogs have a novel role in regulating DNA repair by non-homologous end joining (NHEJ). Here we summarize our findings and present mechanistic hypotheses on how RB1 may support the DNA repair process and the therapeutic implications for patients who harbor RB1-negative cancers.

The Evolutionarily Conserved C-terminal Domains in the Mammalian Retinoblastoma Tumor Suppressor Family Serve as Dual Regulators of Protein Stability and Transcriptional Potency

The retinoblastoma (RB) tumor suppressor and related family of proteins play critical roles in development through their regulation of genes involved in cell fate. Multiple regulatory pathways impact RB function, including the ubiquitin-proteasome system with deregulated RB destruction frequently associated with pathogenesis. With the current study we explored the mechanisms connecting proteasome-mediated turnover of the RB family to the regulation of repressor activity. We find that steady state levels of all RB family members, RB, p107, and p130, were diminished during embryonic stem cell differentiation concomitant with their target gene acquisition. Proteasome-dependent turnover of the RB family is mediated by distinct and autonomously acting instability elements (IE) located in their C-terminal regulatory domains in a process that is sensitive to cyclin-dependent kinase (CDK4) perturbation. The IE regions include motifs that contribute to E2F-DP transcription factor interaction, and consistently, p107 and p130 repressor potency was reduced by IE deletion. The juxtaposition of degron sequences and E2F interaction motifs appears to be a conserved feature across the RB family, suggesting the potential for repressor ubiquitination and specific target gene regulation. These findings establish a mechanistic link between regulation of RB family repressor potency and the ubiquitin-proteasome system.