Analysis Of Cell Cycle Regulators Research Articles

Abstract 5634: Tristetraprolin induces an antitumorigenic phenotype in triple negative breast cancer via a novel non-canonical mechanism

Abstract The ultimate cause of death for most triple negative breast cancer (TNBC) patients is metastatic disease, which has a five-year survival rate of 12.8%. This low survival rate is partly due to the lack of targeted treatments for metastatic TNBC, resulting from the heterogeneity of the disease. As such, further research is needed to identify novel molecular mechanisms that can be targeted to suppress TNBC malignancy. Tristetraprolin (TTP) is an RNA-binding protein that binds to AU-rich elements (AREs) in the 3’ UTRs of select mRNAs, including many that encode proteins involved in cancer-related processes, and targets these mRNAs for degradation. Loss of TTP in tumors correlates with increased disease severity and decreased survival, suggesting a role as a tumor suppressor in multiple cancers, including breast cancer. While previous research has shown that TTP can suppress proliferation in breast cancer, the mechanism remains weakly defined. To elucidate how TTP impacts tumorigenic phenotypes in advanced breast cancer, we stably transfected a FLAG-TTP-expressing construct into three aggressive and metastatic TNBC cell lines. We analyzed TTP-induced changes in gene expression patterns among these cell lines using RNA-sequencing and observed that TTP alters the expression levels of many genes. Gene Set Enrichment Analysis (GSEA) of our datasets revealed that TTP affects multiple pathways, including significant suppression of pathways that promote cell growth, various metabolic processes contributing to tumor growth, and the response to hypoxia. As these pathways highly associated with tumorigenesis, we hypothesized that TTP would attenuate multiple tumor phenotypes. Our subsequent functional analyses revealed that TTP potently suppressed four tumor phenotypes in these cell models: cell proliferation, stem cell frequency, migration, and invasiveness. We hypothesized that inhibition of proliferation was mediated by cell cycle alterations, however, flow cytometry analysis revealed no substantial cell cycle perturbations between TTP-expressing cells and controls. Further analysis of cell cycle regulators revealed minimal changes in expression of proteins encoded by known or suspected TTP target mRNAs. Actinomycin D assays surprisingly revealed that TTP had no effect on the decay kinetics of several known TTP targets, suggesting that TTP’s antitumorigenic properties observed in TNBC cells are independent of its RNA-destabilizing function. To confirm this finding, we constructed cell models expressing an RNA-binding defective mutant of TTP, C147R, which recapitulated suppression of the four tumorigenic phenotypes seen with the wild-type protein. Collectively, these findings reveal that expression of TTP can potently suppress several oncogenic phenotypes in TNBC cells via a mechanism independent of its canonical RNA-binding/destabilizing functions. Citation Format: Julia L. Rutherford, Megan B. Stemberger, Rohaan Mahmud, Christina R. Ross, Elizabeth J. White, Gerald M. Wilson. Tristetraprolin induces an antitumorigenic phenotype in triple negative breast cancer via a novel non-canonical mechanism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5634.

PDZD2 Is Essential for Steady-State Hematopoiesis and Its 37-Kda Secreted Product, sPDZD2, Functions As a Soluble Tumor Suppressor in AML

PDZ domain-containing protein 2 (PDZD2) is expressed in normal hematopoietic stem and progenitor cells (HSPC), specifically HSC, MPP and erythroid progenitors, however, it is almost universally lost through hypermethylation in myeloid malignancies. PDZD2 encodes for a 300kDa protein containing 6 PDZ domains. The full-length protein is cleaved in the cytoplasm in a caspase 3-dependent manner, and the C-terminal fragment containing PDZ domains 5 and 6 is secreted to the tissue microenvironment as a 37kDa protein known as secreted PDZD2 (sPDZD2). Given that sPDZD2 has been reported to function as a tumor suppressor in solid tumors and its almost universal loss in AML, we hypothesized that PDZD2 is required for normal hematopoiesis and that sPDZD2 functions as a soluble tumor suppressor in the hematopoietic system. To determine if sPDZD2 is secreted to the bone marrow (BM) microenvironment we analyzed sPDZD2 levels in BM plasma isolated from primary AMLs and healthy donors using an ELISA-based detection method. We confirmed sPDZD2 secretion by normal CD34+ HSPC and detected significantly lower levels in AMLs (169 ± 17.53 vs 46.12 ± 6.70 ng/ml; p<0.0001). In addition, AML cell lines secreted significantly lower levels of sPDZD2 into the culture medium than HSPCs (28.71 ± 1.76 vs 4.62 ± 1.19 ng/mL; p<0.0001). Notably, treatment of a panel of AML cell lines (n=9) and primary human AML cells (n=4) with recombinant sPDZD2 (r-sPDZD2; dose: 100nM-300nM/day) led to growth inhibition (CellTiter-Glo, p<0.05 for all), differentiation (confirmed by upregulation of CD15/CD11b by flow cytometry) and cell cycle arrest in G0/G1. qRT-PCR analysis of cell cycle regulators in MV4-11 cells treated with r-sPDZD2 confirmed upregulation of CDKN2A (4.01-fold) and E2F4 (4.66-fold) and marked downregulation of multiple cyclin genes, including cyclins D and E, compatible with the cell cycle arrest observed by flow cytometry. To identify signaling pathways downstream of sPDZD2, we treated MV4-11 cells with 100nM r-sPDZD2 and performed phospho-kinase protein array followed by immunoblot for validation. In vitro treatment with r-sPDZD2 led to inhibition of ERK1/2 (T202/Y204, T185/Y187) and CREB (S133) phosphorylation, as well as decreased ß-Catenin expression and increased GSK3ß (S9), RSK1/2 (S221/S227) and p53 (S46) phosphorylation. In order to determine whether PDZD2 plays a role in normal hematopoiesis we performed in vitro differentiation in CRISPR-edited human HSPCs. We used a Cy3-labeled recombinant Cas9 protein and specific single guide RNAs (gRNAs) targeted against PDZD2. Cy3-positive cells were then used for in vitro myeloid or erythroid differentiation, and cell surface markers were evaluated by flow-cytometry analysis over a period of 11 days. While PDZD2-edited cells (PDZD2 KO) displayed a minor defect on myeloid differentiation due to insufficient upregulation of CD15, erythropoiesis appears to be more compromised, with a significant failure to adequately upregulate both CD235a (39 vs 15%) and CD71 (58 vs 26%). To determine the role of Pdzd2 in vivo, we took advantage of a Pdzd2Gt/Gt gene-trap knock-out mouse model and performed peripheral blood (PB) and BM analysis. PB counts at 8-weeks of age showed anemia, with a significant decrease in both red blood cell counts (9.04 ± 0.26 vs 7.58 ± 0.47 x106/µL) and hemoglobin levels (14.88 ± 0.46 vs 12.30 ± 0.81 g/dL) (p<0.05 for both), an observation compatible with the impaired erythropoiesis seen in vitro after PDZD2 KO in human HSPCs. In addition, we observed a trend to decreased MEPs and short-term HSCs, in the BM of 12-week-old mice (n=3), though the small size of the cohort prevented robust statistical analysis. In summary, our findings are the first to shed light on a previously unrecognized role for PDZD2 in hematopoiesis. Loss of PDZD2 in HSPC resulted in impaired erythroid differentiation both in vitro and in vivo. Moreover, our findings validate a soluble tumor suppressor role for sPDZD2 in AML, similar to that seen in solid tumors. Importantly, we further demonstrate a potential role for recombinant sPDZD2 as a novel therapeutic approach for AML.

A quantitative and spatial analysis of cell cycle regulators during the fission yeast cycle

We have carried out a systems-level analysis of the spatial and temporal dynamics of cell cycle regulators in the fission yeast Schizosaccharomyces pombe. In a comprehensive single-cell analysis, we have precisely quantified the levels of 38 proteins previously identified as regulators of the G2 to mitosis transition and of 7 proteins acting at the G1- to S-phase transition. Only 2 of the 38 mitotic regulators exhibit changes in concentration at the whole-cell level: the mitotic B-type cyclin Cdc13, which accumulates continually throughout the cell cycle, and the regulatory phosphatase Cdc25, which exhibits a complex cell cycle pattern. Both proteins show similar patterns of change within the nucleus as in the whole cell but at higher concentrations. In addition, the concentrations of the major fission yeast cyclin-dependent kinase (CDK) Cdc2, the CDK regulator Suc1, and the inhibitory kinase Wee1 also increase in the nucleus, peaking at mitotic onset, but are constant in the whole cell. The significant increase in concentration with size for Cdc13 supports the view that mitotic B-type cyclin accumulation could act as a cell size sensor. We propose a two-step process for the control of mitosis. First, Cdc13 accumulates in a size-dependent manner, which drives increasing CDK activity. Second, from mid-G2, the increasing nuclear accumulation of Cdc25 and the counteracting Wee1 introduce a bistability switch that results in a rapid rise of CDK activity at the end of G2 and thus, brings about an orderly progression into mitosis.

Transcription factor NRF2 participates in cell cycle progression at the level of G1/S and mitotic checkpoints.

Transcription factor NRF2 is considered a master regulator of cell homeostasis, coordinating multiple cytoprotective responses that confer growth advantage to cells. Although its implication in redox homeostasis, detoxification or metabolic reprogramming has been widely studied, its participation in cell cycle division and the mechanisms governing this process has not been explored in detail. In this study, we used several standard methods of synchronization of proliferating cells in G1, before S entry and during mitosis. Together with flow cytometry analysis, we monitored the participation of NRF2 along the cell cycle by knockdown of its gene expression. Our results showed that NRF2 levels are maximal at S phase entry, and minimal during mitosis. Besides, NRF2 absence caused both G1 and M arrest. A targeted transcriptomics analysis of cell cycle regulators showed that NRF2 depletion leads to changes in key cell cycle regulators, including CDK2, TFDP1, CDK6, CDKN1A (p21), CDKN1B (p27), CCNG1 and RAD51. This study gives a new dimension to NRF2 effects, showing its implication in cell cycle progression.

Transcription Factor NRF2 Participates in Cell Cycle Progression at the Level of G1/S and Mitotic Checkpoints.

Transcription factor NRF2 is a master regulator of the multiple cytoprotective responses that confer growth advantages on a cell. However, its participation in the mechanisms that govern the cell division cycle has not been explored in detail. In this study, we used several standard methods of synchronization of proliferating cells together with flow cytometry and monitored the participation of NRF2 along the cell cycle by the knockdown of its gene expression. We found that the NRF2 levels were highest at S phase entry, and lowest at mitosis. NRF2 depletion promoted both G1 and M arrest. Targeted transcriptomics analysis of cell cycle regulators showed that NRF2 depletion leads to changes in key cell cycle regulators, such as CDK2, TFDP1, CDK6, CDKN1A (p21), CDKN1B (p27), CCNG1, and RAD51. This study gives a new dimension to NRF2 effects, showing their implication in cell cycle progression.

Tetraspanin CD82 Regulates Hematopoietic Stem and Progenitor Cell Quiescence and Regeneration

The significant cellular demand of the hematopoietic system is maintained by a rare pool of tissue-specific, hematopoietic stem and progenitor cells (HSPCs) that are primarily found in a quiescent state. Upon hemopoietic stresses, such as significant bleeding, overwhelming infection, and myelosuppressive therapy, HSPCs undergo rapid cell cycle activation, but ultimately must return to quiescence to prevent exhaustion of the hematopoietic system. Emerging evidence from our laboratory suggests that the tetraspanin CD82 plays a critical role in the regulation of HSPC quiescence and activation. Tetraspanins are membrane scaffold proteins with the ability to modulate signaling through the formation of tetraspanin-enriched microdomains, which organize membrane signaling receptors and intracellular signaling molecules critical for propagating downstream signaling. Previous data from our laboratory identified a role for CD82 in HSPC quiescence, where we find a reduction in long term-HSCs in global CD82KO mice, resulting from increased HSPC activation and a reduction of quiescent G 0 cells. In the present study, we test the hypothesis that CD82 expression promotes HSPC return to quiescence following hematopoietic stress, by regulating the activation of TGF-β signaling. To investigate the impact of CD82 expression on hematopoietic regeneration under stress, we treated WT and CD82KO mice with 2 doses of 200mg/kg of chemotherapy agent 5-FU and measured overall survival. Interestingly, we find that CD82KO mice have significantly longer overall survival compared to their WT counterparts. Moreover, we find an increase in peripheral blood HSPCs in CD82KO mice during the early recovery period from 5FU treatment suggestive of enhanced cell activation upon stress and effective regeneration post injury. Mechanistically, the multifunctional cytokine TGF-β plays an essential role in supporting HSPC quiescence and activation, as one of the most potent inhibitors of HSPC growth both in vitroand in vivo. To investigate whether CD82 plays a role in TGF-β signaling, we stimulated WT and CD82KO HSPCs with TGF-β and measured nuclear translocation of SMAD2/3, a downstream effector of TGF-β activation. Confocal imaging demonstrates that CD82KO HSPCs have decreased nuclear translocation of SMAD2/3 upon TGF-β activation, consistent with reduced TGF-β signaling. Similarly, primary human CD34 +HSPCs sorted for low and high CD82 expression also have decreased and increased SMAD2/3 nuclear translocation, respectively. Moreover, gene expression analysis of cell cycle regulators identified the reduced expression of p57, a downstream gene target of TGF-β signaling in HSPCs harvested from CD82KO mice. Currently, we are investigating the impact of the CD82 scaffold on TGF-β signaling of HSPCs localized within the context of the bone marrow niche, by analyzing bone marrow sections from mice injected with HSPC-like cells differentially expressing CD82. Together, these data suggest that CD82 regulates HSPC quiescence and activation through modulation of TGFβ signaling. Future studies will focus on investigating how CD82 modulates local TGFβ signaling within the niche. DisclosuresNo relevant conflicts of interest to declare.

A Novel Phthalimide-Derivative, TC11, Induced G2/M Arrest and Apoptosis of High-Risk Myeloma Cells By Interacting with Nucleophosmin1 and α-Tubulin, but Not By Binding to Celeblon

Background and Purpose:Emergence of novel drugs including immunomodulatory drugs (IMiDs) such as thalidomide, lenalidomide and pomalidomide have markedly improved the survival of multiple myeloma (MM) patients. However, IMiDs have substantial limitation in the treatment of MM; little effect on MM patients with high-risk cytogenetic changes and their teratogenicity. Recently it was reported that IMiDs showed anti-tumor activity as well as teratogenicity by directly binding to cereblon (CRBN).We have previously reported that a novel phthalimide-derivative, 2-(2,6-diisopropylphenyl)-5-amino-1H-isoindole-1,3-dione (TC11), induced apoptosis of high-risk MM cells in vivo and also showed anti-osteoclastogenic activity. At the same time, we observed abnormal α-tubulin fragmentation in TC11-treated MM cell (Matsushita et. al. PLoS One 2015). We have also demonstrated that TC11 bound to nucleophosmin 1(NPM1) and α-tubulin using in vitro virus (IVV) method (Shiheido et. al. PLoS One 2012). Interestingly, lenalidomide also bound to NPM1. The purpose of this study is to clarify molecular mechanisms of anti-myeloma effects of TC11.Results and Discussion:(1) Effect of TC11 is CRBN-independent:Recently it was reported that lenalidomide bound to CRBN, resulting in degradation of its substrates, IKZF1/3 and CK1α. In this study, we found that lenalidomide caused degradation of IKZF1/3 and CK1α in the high-risk MM cell line, KMS34. However, TC11 didn't down-regulate CRBN or its substrates, IKZF1/3 and CK1α. Taken together with the result of Biacore assay, TC11 induced MM cell death via CRBN-independent pathway.(2) Cell cycle arrest by TC11 vs. lenalidomide:Then we tried to elucidate molecular mechanisms of TC11 by the microarray analysis of KMS21, which is highly sensitive to TC11 and lenalidomide. Microarray Gene Set Enrichment Analysis (GSEA) indicated that TC11 upregulated the gene set of G2/M checkpoint, however lenalidomide did not. These results were also confirmed by analyzing the DNA contents of MM cells using flow cytometry and by western blot analysis of cell cycle regulators, phosphorylated cdc2 and p21. These data suggest that TC11 induced apoptosis of MM cells by distinct molecular mechanism from lenalidomide probably by regulating G2/M cell cycle. TP53 gene regulates G1/S cell cycle and occasionally deleted in high-risk MM cells. However, TC11 is able to induce apoptosis of TP53 -deleted high-risk MM cells by causing G2/M arrest.(3) Inhibition of tubulin assembly by TC11:Since we have previously showed that TC11 bound to α-tubulin by IVV method, we speculated that TC11 affected tubulin polymerization or depolymerization. Treatment of MM cells with TC11 significantly reduced polymerized fraction of α-tubulin, indicating TC11 inhibited tubulin polymerization. In contrast, lenalidomide did not inhibit either tubulin polymerization or depolymerization. In this regard, the mechanism of TC11 is different from that of lenalidomide.(4) Inhibition of oligomerization of NPM1 by TC11:We have also showed that TC11 bound to NPM1 by IVV method (Shiheido et. al. PLoS One 2012). It has been reported that oligomerized NPM1 had pleiotropic biological functions. Thus we examined whether TC11 affected NPM1 oligomerization using blue native polyacrylamide gel electrophoresis. As a result, TC11 treatment significantly reduced the amount of oligomerized NPM1 fraction. At the same time, we found that TC11 significantly increased in phosphorylation of NPM1 at Ser4, Thr95 and Thr199. It has been reported that phosphorylation of NPM1 inhibited its oligomerization (Mitrea et. al. Proc Natl Acad Sci U S A 2014). Thus, we speculated that TC11-induced phosphorylation resulted in inhibition of oligomerization of NPM1.Conclusion:It was shown that TC11 bound to α-tubulin and NPM1, but not to CRBN. TC11 inhibited tubulin assembly as well as NPM1 oligomerization, leading to G2/M arrest and mitotic catastrophe. These novel molecular events by TC11 induced apoptosis of TP53 -deleted high-risk MM cells. [Display omitted] DisclosuresYonemura:IDAC Theranostics Inc.: Employment. Tabata:IDAC Theranostics Inc.: Employment. Yanagawa:IDAC Theranostics Inc.: Employment. Hattori:Cosmic Corporation Co., Ltd.: Research Funding; Merck Sharp & Dohme: Research Funding; Takeda Pharmaceutical Company Limited.: Research Funding.

Genetic Association Analysis of Cell Cycle Regulators Reveals YWHAZ Has Prognostic Significance in Prostate Cancer.

This study aimed to identify the genes that cause biochemical recurrence (BCR) following radical prostatectomy (RP) in men with localized prostate cancer. A two-stage genetic association study of 19 single-nucleotide polymorphisms in 11 key cell cycle regulation genes was carried out. BCR-free survival after RP was evaluated in a discovery cohort of 458 patients with prostate cancer, and replication was investigated in another cohort of 185 patients. A consistent association was found between BCR and rs2290291 (discovery: p=0.008; replication: p=0.029). rs2290291 is located in the tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ), and was predicted to possess a regulatory function that affected YWHAZ expression. Furthermore, YWHAZ expression was frequently up-regulated in advanced tumours, and associated with poorer survival in patients with prostate cancer. YWHAZ rs2290291 was found to be associated with BCR. YWHAZ may function as a putative oncogene during prostate cancer progression.

APC/C is essential for hematopoiesis and impaired in aplastic anemia.

Anaphase promoting complex/cyclosome (APC/C) is essential for cell cycle progression. Recently, its non-mitotic functions were also reported but less studied in several tissues including hematopoietic cells. Here, we developed an inducible Anapc2 (a core subunit of APC/C) knockout mice. The animals displayed a fatal bone marrow failure within 7 days after knockout induction. Their hematopoietic stem and progenitor cells (HSPCs) demonstrated a sharp decline and could form little colony. Further, the results of BrdU label-retaining cell assay showed that the dormant HPSCs lost rapidly. Analysis of cell cycle regulators, Skp2, P27, Cdk2, and Cyclin E1, suggested that these quiescent stem cells underwent a shift from quiescence to mitosis followed by apoptosis. We next detected Anapc2-expression in the CD34+ HSPCs of patients with aplastic anemia. CD34+ cells were markedly decreased in the bone marrow and Anapc2-expression in the residual CD34+ cells was undetectable, suggesting that APC/C was deficient and might have a relationship with the pathogenesis of aplastic anemia.

Magnolol Inhibits the Growth of Non-Small Cell Lung Cancer via Inhibiting Microtubule Polymerization

Background: The tubulin/microtubule system, which is an integral component of the cytoskeleton, plays an essential role in mitosis. Targeting mitotic progression by disturbing microtubule dynamics is a rational strategy for cancer treatment. Methods: Microtubule polymerization assay was performed to examine the effect of Magnolol (a novel natural phenolic compound isolated from Magnolia obovata) on cellular microtubule polymerization in human non-small cell lung cancer (NSCLC) cells. Cell cycle analysis, mitotic index assay, cell proliferation assay, colony formation assay, western blotting analysis of cell cycle regulators, Annexin V-FITC/PI staining, and live/dead viability staining were carried out to investigate the Magnolol’s inhibitory effect on proliferation and viability of NSCLS cells in vitro. Xenograft model of human A549 NSCLC tumor was used to determine the Magnolol’s efficacy in vivo. Results: Magnolol treatment effectively inhibited cell proliferation and colony formation of NSCLC cells. Further study proved that Magnolol induced the mitotic phase arrest and inhibited G2/M progression in a dose-dependent manner, which were mechanistically associated with expression alteration of a series of cell cycle regulators. Furthermore, Magnolol treatment disrupted the cellular microtubule organization via inhibiting the polymerization of microtubule. We also found treatment with NSCLC cells with Magnolol resulted in apoptosis activation through a p53-independent pathway, and autophgy induction via down-regulation of the Akt/mTOR pathway. Finally, Magnolol treatment significantly suppressed the NSCLC tumor growth in mouse xenograft model in vivo. Conclusion: These findings identify Magnolol as a promising candidate with anti-microtubule polymerization activity for NSCLC treatment.

Abstract 1854: Evidence that RARs interact with Src family kinases and that inhibition of Src family kinase activity can affect the growth response of SKOV3 ovarian cancer cell to atRA

Abstract Chemotherapy resistance is a major cause of ovarian cancer treatment failure. All trans-retinoic acid (atRA) is a promising therapeutic agent due to its growth suppressing activity however, not all ovarian carcinomas are growth inhibited by atRA. Understanding the underlying mechanisms of atRA action in ovarian cancer is crucial to overcome chemotherapy resistance. Recent studies have reported novel nongenomic actions of atRA and retinoic acid receptors (RARs). The main goal of this work was to study nongenomic actions of RARs. A SH3 domain phage display was performed to identify proteins that interact with the proline rich region of RARγ. Our results indicated that RARγ proline rich region binds to the SH3 domain of Src family kinase proteins (SFKs) Lyn, Src and Yes. Immunofluorescence assays showed that RARγ colocalizes in the cell cytoplasm with Lyn, Src and Yes. Proximity ligation assays were performed to determine whether RARs and Lyn, Src and Yes interact in vivo. Our results showed that RARα and RARγ but not RARβ form a complex with both Lyn and Src. No interaction was found between any of the RARs and Yes. Interaction between RARs and SFKs was found to be directly correlated with phosphorylation of Y419 in Src and Y397 in Lyn. We have previously shown that the ovarian cancer cell line CAOV3 is sensitive to growth inhibition by atRA while the ovarian cancer cell line SKOV3 is resistance to atRA growth repression. Since SFKs such as Src and Lyn are known to regulate cancer cell growth, we next investigated the effect of inhibiting SFKs activity on the atRA regulated growth response of these cell lines. CAOV3 and SKOV3 cells were treated with atRA (1μM) alone or in combination with varying doses of SFKs inhibitors PP1 and PP2 and analyzed for cell growth inhibition. CAOV3 but not SKOV3 cells were growth arrested by atRA. Both PP1 and PP2 caused growth inhibition in both cell lines. Interestingly, when atRA was combined with either of the SFKs inhibitors, SKOV3 but not CAOV3 cells exhibited growth inhibition greater than that observed with PP1 or PP2 alone. These data demonstrate that inhibition of SFKs activity converts the growth response of SKOV3 cells from atRA resistant to atRA sensitive. Analysis of cell cycle regulators in atRA and PP1 treated CAOV3 cells showed that both p27 and p130 are up-regulated while CDK6 is down-regulated. On the other hand, in SKOV3 cells treated with atRA and PP1, p130 is unaffected while p27, cyclin A, CDK1, CDK2 and CDK6 are down-regulated. In conclusion, we have confirmed the interaction between RARγ and Src and describe for the first time the interaction between RARα and both Lyn and Src, and RARγ and Lyn. We also provide evidence that reduction of SFKs activity and atRA treatment but not atRA treatment alone can inhibit the growth of SKOV3 cells. This suggests that SFKs activity must be reduced in order for atRA to inhibit cell growth in at least some ovarian carcinomas. Citation Format: Thais Acquafreda Lakind, Kenneth Soprano, Dianne Soprano. Evidence that RARs interact with Src family kinases and that inhibition of Src family kinase activity can affect the growth response of SKOV3 ovarian cancer cell to atRA. [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 1854. doi:10.1158/1538-7445.AM2015-1854

Preliminary analysis of cell cycle regulation in cardiomyocytes.

Preliminary analysis of cell cycle regulation in cardiomyocytes.

Combination of immortalization and inducible death strategies to generate a human mesenchymal stromal cell line with controlled survival

The hTERT-immortalization of human bone marrow-derived Mesenchymal Stromal Cells (hMSCs) was proposed to address availability/standardization issues for experimental or clinical studies, but raised concerns due to possible uncontrolled growth or malignant cell transformation.Here we report a method to generate a hMSCs line with controlled survival, through the implementation of a pre-established suicide system (inducible caspase 9, iCasp9) in hTERT-transduced hMSCs.Primary hMSCs were successfully immortalized (>280 PD) and further transduced with the iCasp9 device. A clone was selected and shown to maintain typical properties of primary hMSCs, including phenotype, differentiation and immunomodulation capacities. The successive transductions did not induce tumorigenic transformation, as assessed by analysis of cell cycle regulators and in vivo luciferase-based cell tracking. Cells could be efficiently induced toward apoptosis (>95%) both in vitro and in vivo.By combining the opposite concepts of ‘induced-life’ and ‘inducible-death’, we generated a hMSCs line with defined properties and allowing for temporally controlled survival. The cell line represents a relevant tool for medical discovery in regenerative medicine and a potential means to address availability, standardization and safety requirements in cell & gene therapy. The concept of a hTERT-iCasp9 combination, here explored in the context of hMSCs, could be extended to other types of progenitor/stem cells.

Abstract 255: Expressionprofiling of stem cell signaling alters with spheroid formation in CD133high/CD44high prostate cancer stem cells.

Abstract Introduction: Cancer stem cell (CSCs) theory provides new insight into the formation of tumors. Characterization of the pathways which regulate CSCs activity will facilitate the development of targeted therapies. Isolation of CSCs from multiple tumor types differentiate both in vivo and in vitro when cultured in serum, yet the factors responsible for their differentiation have not yet been identified. Current challenge is the enrichment of CSCs from established cell lines variety of solid tumors has been developed as three-dimensional (3D) cell culture. 3D spheroid model is a new technique for propagation of cells in-vitro using serum free medium and cultured low-adherence conditions and present a convenient model to investigate differentiating cancer cells. The objective of this study is to identify CD 133high/ CD44 high prostate CSCs and compare these profiles with non-sorting cells as bulk counterparts of the population. These two populations have been continued to be 3D multicellular spheroids and differentiation has been investigated with stem cell related genomic characteristics. Since CSCs are proliferate in a trice in tumor mass, our aim is to reveal the changes of cellular profile and heterogeneity of proliferated cells in 3D multicellular tumor spheroids. Material and method: CSCs and non- CSCs were sorted with FACS and PCR array analysis of cell cycle regulation, embryonic and mesenchymal cell lineage related markers, TERT and Notch signaling were performed in both monolayer and spheroids. Immunohistochemistry of CD117, Notch1, Jagged1, Delta1, Sox2, C-myc, Oct4, KLF4, CD90 and SSEA1 were determined in groups. Results and discussion: Most significant gene alteration observed CD 133high/ CD44 high population when in monolayer cells continued as spheroid. In this group remarkable up regulation was determined in Collagen type 9 alpha1 (COL9A1), Islet1 (ISL1) and Cyclin D2. Jagged1, DLL3 and Notch1 were respectively up-regulated genes in Notch signaling. According to immunoreactivity staining density of Jagged1, Sox2, Oct4 and Klf-4 increased significantly in sorting spheroids when compared non-sorting monolayer cells. Conclusion: Isolated CSCs in human tumors could be altered their cellular characterization in the course of time and display a differentiation with maintaining their former surface antigens at a level of transcription or translation. In this study we have indicated this differentiation theory by using in-vitro 3D- spheroid model. It was observed that the cells alter their gene expression profiles during this process. Our hypothesis was supported by the observation that most significant gene alteration was in CD 133high/ CD44 high population when monolayer cells were allowed to grow as spheroid. In fact, this differentiation could be a principal mechanism which is responsible from malign process and tumor growth. Citation Format: Gulperi Oktem, Ayhan Bilir, Ruchan Uslu, Sevinc Inan, Sirin Bakti Demiray, Harika Atmaca, Sule Ayla, Aysegul Uysal. Expressionprofiling of stem cell signaling alters with spheroid formation in CD133high/CD44high prostate cancer stem cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 255. doi:10.1158/1538-7445.AM2013-255

Cell cycle regulation in mouse heart during embryonic and postnatal stages

The regulation of cardiomyocyte proliferation is important for heart development and function. Proliferation levels of mouse cardiomyocytes are high during early embryogenesis and start to decrease at midgestation. Many cardiomyocytes undergo mitosis without cytokinesis, resulting in binucleated cardiomyocytes during early postnatal stages, following which the cell cycle arrests irreversibly. It remains unknown how the proliferation pattern is regulated, and how the irreversible cell cycle arrest occurs. To clarify the mechanisms, fundamental information about cell cycle regulators in cardiomyocytes and cell cycle patterns during embryonic and postnatal stages is necessary. Here, we show that the expression, complex formation, and activity of main cyclins and cyclin-dependent kinases (CDKs) changed in a synchronous manner during embryonic and postnatal stages. These levels decreased from midgestation to birth, and then showed one wave in which the peak was around postnatal day 5. Detailed analysis of the complexes suggested that CDK activities were inhibited before the protein levels decreased. Analysis of DNA content distribution patterns in mono- and binucleated cardiomyocytes after birth revealed changes in cell cycle distribution patterns and the transition from mono- to binucleated cells. These analyses indicated that the wave of cell cycle regulator expression or activities during postnatal stages mainly produced binucleated cells from mononucleated cells. The data obtained should provide a basis for the analysis of cell cycle regulation in cardiomyocytes during embryonic and postnatal stages.

A Systematic Analysis of Cell Cycle Regulators in Yeast Reveals That Most Factors Act Independently of Cell Size to Control Initiation of Division

Upstream events that trigger initiation of cell division, at a point called START in yeast, determine the overall rates of cell proliferation. The identity and complete sequence of those events remain unknown. Previous studies relied mainly on cell size changes to identify systematically genes required for the timely completion of START. Here, we evaluated panels of non-essential single gene deletion strains for altered DNA content by flow cytometry. This analysis revealed that most gene deletions that altered cell cycle progression did not change cell size. Our results highlight a strong requirement for ribosomal biogenesis and protein synthesis for initiation of cell division. We also identified numerous factors that have not been previously implicated in cell cycle control mechanisms. We found that CBS, which catalyzes the synthesis of cystathionine from serine and homocysteine, advances START in two ways: by promoting cell growth, which requires CBS's catalytic activity, and by a separate function, which does not require CBS's catalytic activity. CBS defects cause disease in humans, and in animals CBS has vital, non-catalytic, unknown roles. Hence, our results may be relevant for human biology. Taken together, these findings significantly expand the range of factors required for the timely initiation of cell division. The systematic identification of non-essential regulators of cell division we describe will be a valuable resource for analysis of cell cycle progression in yeast and other organisms.

Dedifferentiated peripheral chondrosarcoma: a clinicopathologic, immunohistochemical, and molecular analysis of four cases

Peripheral dedifferentiated chondrosarcoma (DCS) is an exceedingly rare aggressive surface bone neoplasm in which a high-grade sarcoma arises within an osteochondroma. Four such examples were identified in our files, representing 11.1% of all DCS treated at our hospital in the years 1995-2010, and were the object of the present study. The patients were two men and two women ranging in age between 30 and 64 years, with tumors located in the pelvis (n = 2), in the scapula (n = 1), and the tibia (n = 1). Radiologically, there was evidence of a preexisting osteochondroma associated with aggressive osteolytic areas at the base and periphery of the exostosis, extending to the bone segment of origin and to the soft tissues. Immunohistochemical analysis of cell cycle regulators showed consistent loss in the expression of p16 and overexpression of cyclin D1, and to a lesser extent of RB and p53, in the anaplastic compartments of peripheral DCS in comparison with the well-differentiated cartilaginous components, while no significant expression of MDM2 was observed in any of the cases studied. Moreover, PDGFRα was absent in both tumor components, and PDGF-Rβ was strongly and diffusely positive in all the cases. Finally, no mutations were identified in exons 4-9 of the TP53 gene in both cases, showing positivity for p53 in the anaplastic component. In conclusion, our study shows that alterations of genes implicated in the regulation of the G1 to the S phase cell cycle checkpoint contribute to the process of dedifferentiation in peripheral secondary chondrosarcoma (CS), although the molecular mechanisms seem at least in part to differ from those involved in the process of dedifferentiation of central CS. PDGFRβ could represent a potential target for treatments with receptor tyrosine kinase inhibitors in peripheral DCS.

EphrinB1 Activation As a Potential New Treatment Option in AML

Abstract 5235Aberrant Ephrin signaling has been shown to be an important pathway that contributes to the pathogenesis of many solid tumors (Surawska et al. Cytokine & Growth factor reviews 2004). Deregulated ephrin receptor (Eph) and ligand (Efn) expression is often associated with poor prognosis in solid tumors. Ephrin receptor and ligand overexpression can result in tumorigenesis through induced tumor growth, tumor cell survival, angiogenesis and metastasis (Surawska et al. Cytokine & Growth Factor Reviews 2004; Campbell et al. Curr. Isues Mol. Biol. 2008; Chen et al. Cancer Research 2008). In normal cells Eph receptors and ligands play key roles in vascular patterning, where they function in endothelial cell migration, and proliferation (Adams et al, Genes Dev. 1999; Zhang et al., Blood 2001). Thus far particularly EphB4 receptor and ephrin-B2 ligand have been implicated in the process of normal angiogenesis. In acute myeloid leukemia (AML) patients it was found that bone marrow biopsies at diagnosis exhibited enhanced microvessel density (MVD) (de Bont ES et al., BJH 2001; Byrd JC et al., Blood 2002; Padro et al., Blood 2000). Normal hematopoietic stem cells (HSCs) express the following mRNA transcripts ephrin receptors EphA1, EphA2, EphB2, and EphB4 and ephrin ligands EfnA3, EfnA4, and EfnB2. Moreover, overexpression of EphB4 receptor in HSCs (from cord blood) resulted in enhanced differentiation towards megakaryocytes (Wang et al. Blood 2002). In AML cell lines there is a common co-expression on protein level observed between EphB4 receptor and ephrin-B2 ligand. Recently, an aberrant DNA methylation of ephrin receptors and ligands was described in acute lymphocytic and myelocytic leukemia cell lines (Kuang et al. Blood 2010). In addition, restoration of EphB4 expression in an acute lymphoid leukemia cell line resulted in reduced proliferation and apoptotic cell death. These data suggests that the ephrin signaling pathway might play an important role in leukemia. In a previous study we have found high kinase activity of EphB receptors and high phosphorylation levels of EphB receptors in AML samples, as measured using kinase arrays and proteome profiler arrays. In this study, we have found extensive membrane expression of EphB1 on AML cell lines and primary AML blasts. To identify the role of Ephrin signaling in AML, two AML cell lines THP-1 and HL60 with an EphB1 membrane expressing cell percentage of 70% and 20% respectively were chosen for stimulation with Ephrin-B1 ligand. Treatment of these cell lines with Ephrin-B1 ligand resulted in a decreased proliferation 30% in THP-1 cells versus 22% in HL60 cells and increased apoptosis 23% in THP-1 cells and 4% in HL60 cells. Of note, the most prominent effect of Ephrin-B1 stimulation was found in THP-1 cells, this cell line contained a higher percentage of EphB1 membrane expressing cells. We further investigated the mechanism through which EphB1 reduces leukemic cell growth and induces leukemic cell death in THP-1 cells. Westernblot analysis of cell cycle regulators showed that expression of the anti-apoptotic protein BCL2 is reduced upon Ephrin-B1 ligand stimulation and the expression of the pro-apoptotic protein BAX is induced. In addition, mRNA expression of the cell cycle inhibitor of cell cycle progression p21 was found to be 2,5 fold upregulated in ephrin-B1 ligand treated cells compared to untreated control cells. MGG stainings of Ephrin-B1 treated cells revealed multiple cells with two nuclei in both THP-1 and HL60 cells. These results indicate that a high percentage of AML cells express EphB1 receptor on the membrane and that stimulation of these cells with Ephrin-B1 ligand results in reduced leukemic growth and increased cell death. EphrinB1 activation in AML deserves further investigation considering EphB1 as a putative new treatment option for AML patients. Disclosures:No relevant conflicts of interest to declare.

Knockdown of Fbxo7 reveals its regulatory role in proliferation and differentiation of haematopoietic precursor cells

Fbxo7 is an unusual F-box protein because most of its interacting proteins are not substrates for ubiquitin-mediated degradation. Fbxo7 directly binds p27 and Cdk6, enhances the level of cyclin D-Cdk6 complexes, and its overexpression causes Cdk6-dependent transformation of immortalised fibroblasts. Here, we test the ability of Fbxo7 to transform haematopoietic pro-B (Ba/F3) cells which, unexpectedly, it was unable to do despite high levels of Cdk6. Instead, reduction of Fbxo7 expression increased proliferation, decreased cell size and shortened G1 phase. Analysis of cell cycle regulators showed that cells had decreased levels of p27, and increased levels of S phase cyclins and Cdk2 activity. Also, Fbxo7 protein levels correlated inversely with those of CD43, suggesting direct regulation of its expression and, therefore, of B cell maturation. Alterations to Cdk6 protein levels did not affect the cell cycle, indicating that Cdk6 is neither rate-limiting nor essential in Ba/F3 cells; however, decreased expression of Cdk6 also enhanced levels of CD43, indicating that expression of CD43 is independent of cell cycle regulation. The physiological effect of reduced levels of Fbxo7 was assessed by creating a transgenic mouse with a LacZ insertion into the Fbxo7 locus. Homozygous Fbxo7(LacZ) mice showed significantly increased pro-B cell and pro-erythroblast populations, consistent with Fbxo7 having an anti-proliferative function and/or a role in promoting maturation of precursor cells.

Knockdown of Fbxo7 reveals its regulatory role in proliferation and differentiation of haematopoietic precursor cells

Fbxo7 is an unusual F-box protein because most of its interacting proteins are not substrates for ubiquitin-mediated degradation. Fbxo7 directly binds p27 and Cdk6, enhances the level of cyclin D-Cdk6 complexes, and its overexpression causes Cdk6-dependent transformation of immortalised fibroblasts. Here, we test the ability of Fbxo7 to transform haematopoietic pro-B (Ba/F3) cells which, unexpectedly, it was unable to do despite high levels of Cdk6. Instead, reduction of Fbxo7 expression increased proliferation, decreased cell size and shortened G1 phase. Analysis of cell cycle regulators showed that cells had decreased levels of p27, and increased levels of S phase cyclins and Cdk2 activity. Also, Fbxo7 protein levels correlated inversely with those of CD43, suggesting direct regulation of its expression and, therefore, of B cell maturation. Alterations to Cdk6 protein levels did not affect the cell cycle, indicating that Cdk6 is neither rate-limiting nor essential in Ba/F3 cells; however, decreased expression of Cdk6 also enhanced levels of CD43, indicating that expression of CD43 is independent of cell cycle regulation. The physiological effect of reduced levels of Fbxo7 was assessed by creating a transgenic mouse with a LacZ insertion into the Fbxo7 locus. Homozygous Fbxo7(LacZ) mice showed significantly increased pro-B cell and pro-erythroblast populations, consistent with Fbxo7 having an anti-proliferative function and/or a role in promoting maturation of precursor cells.