Regulation Of Cell Cycle Control Research Articles

Mechanism of activating mutations and allosteric drug inhibition of the phosphatase SHP2

Protein tyrosine phosphatase SHP2 functions as a key regulator of cell cycle control, and activating mutations cause several cancers. Here, we dissect the energy landscape of wild-type SHP2 and the oncogenic mutation E76K. NMR spectroscopy and X-ray crystallography reveal that wild-type SHP2 exchanges between closed, inactive and open, active conformations. E76K mutation shifts this equilibrium toward the open state. The previously unknown open conformation is characterized, including the active-site WPD loop in the inward and outward conformations. Binding of the allosteric inhibitor SHP099 to E76K mutant, despite much weaker, results in an identical structure as the wild-type complex. A conformational selection to the closed state reduces drug affinity which, combined with E76K’s much higher activity, demands significantly greater SHP099 concentrations to restore wild-type activity levels. The differences in structural ensembles and drug-binding kinetics of cancer-associated SHP2 forms may stimulate innovative ideas for developing more potent inhibitors for activated SHP2 mutants.

Ceramide synthase 6 predicts the prognosis of human gastric cancer: It functions as an oncoprotein by dysregulating the SOCS2/JAK2/STAT3 pathway.

Although gastric cancer (GC) is one of the most common cancers, knowledge of its development, and carcinogenesis is limited. The present study explored the involvement of ceramide synthase 6 (CERS6) in GC carcinogenesis and prognosis. RT-PCR, immunoblotting, and immunohistochemistry were used to examine the expression of CERS6. Transfection and small hairpin RNA technology were used to investigate the effect of CERS6 manipulation on cell proliferation and spread as well as the underlying mechanism. Moreover, xenograft proliferation was employed to explore the influence of CERS6 on tumor growth in animals. It was found that overexpression of CERS6 was significantly correlated with several clinicopathologic parameters and poor disease-free survival. The overexpression and silencing of CERS6 in GC cells facilitated and suppressed cell proliferation and spread as well as xenograft proliferation, respectively. Mechanistic studies further revealed that CERS6 influenced cell proliferation and spread by regulating cell cycle control and metastasis-related protein through the SOCS2/JAK2/STAT3 signaling pathway. Collectively, this study suggests that CERS6 overexpression could be a useful biomarker for predicting the outcomes of GC patients and that CERS6 targeting represents a potential modality for treating GC.

Samul-Tang Regulates Cell Cycle and Migration of Vascular Smooth Muscle Cells against TNF-α Stimulation.

Samul-Tang (SMT), consisting of four medicinal herbs, is a well-known herbal prescription treating hematological disorders related symptoms. Our previous study demonstrated that SMT attenuated inflammation of vascular endothelial cells. In condition of retained vascular dysfunction, vascular inflammation is initiated and results in activation of smooth muscle cells (SMCs). Activated SMCs lose control of cell cycle regulation and migrate into intima, resulting in formation of atheroma. Here, we further investigated whether SMT suppresses proliferation and migration of SMCs. SMT showed antiproliferative effects on SMCs by suppressing [3H]-thymidine incorporation against TNF-α stimulation. Underlying mechanisms of antiproliferative effects were found to be resulting from cell cycle regulation. SMT downregulated expression of cyclin D1-CDK4 and cyclin E-CDK2 complexes and upregulated p21waf1/cip1 and p27kip1. SMT also suppressed migration of SMCs against TNF-α stimulation. This is thought to have resulted from suppressing MMP2 and MMP9 expressions and ROS production. In summary, SMT attenuates abnormal migration of vascular smooth muscle cells via regulating cell cycle and suppressing MMPs expression and ROS production. Our study suggests that SMT, a traditionally used herbal formula, protects vascular smooth muscle cells and might be used as an antiatherosclerotic drug.

Abstract 45: BTM-3528 potently induces G1/G0 cell cycle arrest and is efficacious in preclinical models of diffuse large B-cell lymphoma

Abstract Aberrant regulation of cell cycle control, integration of signals from oncogenic signaling pathways, and cancer cell metabolism play a key role in the pathogenesis of multiple hematologic malignancies and solid tumors. BTM-3528 is an orally available compound that causes concentration-dependent cell cycle arrest in the G0/G1 phase of the cell cycle, rapidly decreases cellular glutathione levels, induces expression of p21, and decreases expression of Cyclin D 1/3. BTM-3528 has no significant biochemical activity against a panel of known drug targets such as kinases (including cyclin-dependent kinases), G-protein coupled receptors, ion channels, and histone deacetylases. For the described study, the activity of BTM-3528 was evaluated in in vitro and in vivo models of diffuse large B-cell lymphoma (DLBCL). Profiling of BTM-3528 across a panel of hematologic and solid tumor cancer cell lines demonstrated potent antiproliferative activity against a select set of cancer cell types without effect on normal human cells. These included multiple DLBCL cell lines notably of germinal center B-cell (GCB) subtype. The majority of the DLBCL cell lines tested were characterized by Myc and Bcl-2 gene rearrangements or amplifications. Cell proliferation was 100% inhibited with IC50s ranging from 0.14 to 0.52 μM in these DLBCL cell lines. BTM-3528 induced apoptosis with caspase 3/7 activation and decreased Mcl1 protein expression at concentrations associated with cell growth inhibition. In vivo pharmacokinetic studies showed that BTM-3528 has excellent oral bioavailability with a murine t ½ of 6 hours. BTM-3528 was tested in a human subcutaneous DLBCL xenograft model (BJAB). Post tumor implantation, animals with tumors ≥ 250 mm3 were treated with BTM-3528 at the maximum tolerated dose of 75 mg/kg once daily orally for 14 days. Significant antitumor activity was observed after 3 days of treatment followed by sustained tumor regressions. Furthermore, following discontinuation of BTM-3528 after 14 days of treatment, sustained complete tumor regressions were maintained in 50% of the treated animals. Preliminary toxicology evaluation of BTM-3528 showed no adverse effect on hematopoiesis or hepatic function. Conclusion: BTM-3528 is a novel orally bioavailable compound with a unique mechanism of action based on G1/G0 cell cycle arrest and modulation of cancer cell metabolism. It shows significant activity and a favorable toxicity profile in in vitro and in vivo models of DLBCL. These results support the clinical development potential of BTM-3528 for the treatment of DLBCL. Citation Format: Matthew Kostura, Jedd Levine, Vibha Oza, Alan Cooper, Meztli Arguello, Peter W. White, Stephane Ciblat, Anthony Regina, Edith Bellavance, Martine Brault, Arshad Siddiqui, Michael Luther. BTM-3528 potently induces G1/G0 cell cycle arrest and is efficacious in preclinical models of diffuse large B-cell lymphoma [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 45.

Pharmacological and transcriptional inhibition of the G9a histone methyltransferase suppresses proliferation and modulates redox homeostasis in human microvascular endothelial cells

Epigenetic mechanisms, including histone post-translational modifications, are central regulators of cell cycle control. The euchromatic G9a histone methyltransferase (G9a HMT) is a key enzyme catalyzing histone H3 methylation on lysines 9 and 27, and its dysregulation has been linked to uncontrolled proliferation of tumor cells. Here, we have investigated the effect of G9a HMT silencing on cell proliferation of microvascular endothelial cells, a process necessary to sustain tumor growth through the formation of the vascular capillary network.Inhibition of G9a HMT activity in human microvascular endothelial cells (HMEC-1) was performed either pharmacologically, by treatment of cells with BIX-01294 or chaetocin, or transcriptionally, using shRNA. Cell viability and proliferation were examined using the resazurin reduction assay, flow cytometry and immunostaining of phosphorylated checkpoint kinase 1 (pSer317Chk1). Expression of cell cycle- and redox homeostasis-related genes was determined by quantitative PCR. Reactive oxygen species production was measured by oxidation of the fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate and the cell’s total antioxidant capacity by using the ABTS assay.Inhibition of G9a HMT activity by BIX-01294 treatment or by shRNA attenuated the proliferation of HMEC-1, nuclear localization of phosphorylated Chk1, and induced cell cycle arrest in G1 phase. Transcriptional analysis demonstrated increased gene expression of the cyclin-dependent kinase (CDK) inhibitor p21, and also of Rb1, in BIX-01294 treated cells. Decreased proliferation rate was accompanied by enhanced antioxidant potential of HMEC-1 cells, as demonstrated by reduced production of reactive oxygen species, increased total antioxidant capacity and expression of the antioxidant enzymes catalase and superoxide dismutase 1.Collectively, our results demonstrate of the central role of G9a HMT in the promotion of endothelial cells proliferation, and suggest that endothelial G9a HMT may be a target in the treatment of vascular proliferative disorders and tumor neovascularization.

Abstract 5542: Regulation of cell cycle control in T-cell acute lymphoblastic leukemia by Ikaros and Casein Kinase II

Abstract T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy that represents a therapeutic challenge. Next-generation sequencing revealed that a subset of T-ALL harbors inactivating mutations or deletion of one allele of the IKZF1 tumor suppressor. These data suggest that IKZF1 acts as a tumor suppressor in T-ALL. The IKZF1 gene encodes the Ikaros protein that functions as a regulator of transcription and a tumor suppressor in B cell acute lymphoblastic leukemia. However, the molecular mechanism of Ikaros tumor suppressor function in T-ALL is unclear. Using quantitative chromatin immunoprecipitation (qChIP), we determined that Ikaros binds to the promoter regions of the CDC2 and CDC7 cell cycle genes in primary T-ALL cells in vivo. Gain-of function experiments showed that Ikaros overexpression in T-ALL results in reduced expression of CDC2 and CDC7, as evidenced by quantitative RT-PCR (qRT-PCR) and Western blot. The knock-down of Ikaros with shRNA in T-ALL cells resulted in increased transcription of CDC2 and CDC7 as indicated by qRT-PCR. These data suggest that Ikaros can regulate cell cycle progression in T-ALL by repressing transcription of the CDC2 and CDC7 genes. Next, we studied the mechanisms that regulate Ikaros’ ability to repress CDC2 and CDC7 in T-ALL. Ikaros function as a transcriptional repressor is regulated by Casein Kinase II (CK2). CK2 is overexpressed in hematopoietic malignancies and increased expression of CK2 results in T-ALL in murine models. We tested the effect of CK2 inhibition on Ikaros’ ability to regulate transcription of CDC2 and CDC7 in human T-ALL. Molecular inhibition of CK2 with shRNA against the CK2 catalytic subunit resulted in reduced transcription of CDC2 and CDC7, as evidenced by qRT-PCR. This was associated with increased DNA-binding of Ikaros to promoters of CDC2 and CDC7, as shown by qChIP. These data suggest that CK2 impairs Ikaros’ ability to transcriptionally repress CDC2 and CDC7 and to regulate cell cycle progression in T-ALL. Inhibition of CK2 enhances transcriptional repression of CDC2 and CDC7 by Ikaros, resulting in improved control of cell cycle progression in T-ALL. In conclusion, our results show that control of cell cycle progression in T-ALL occurs trough Ikaros-mediated transcriptional regulation of CDC2 and CDC7. Overexpession of CK2 impairs Ikaros ability to repress CDC2 and CDC7 expression, which contributes to deregulation of cell cycle control in T-ALL. Results suggest a potential mechanism of therapeutic action of CK2 inhibitors for the treatment of T-ALL. Note: This abstract was not presented at the meeting. Citation Format: Mario A. Soliman, Tommy Hu, Malika Kapadia, Elanora Dovat, Yali Ding, Chunhua Song, Jonathon L. Payne, Sinisa Dovat. Regulation of cell cycle control in T-cell acute lymphoblastic leukemia by Ikaros and Casein Kinase II [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5542. doi:10.1158/1538-7445.AM2017-5542

Role of HDM2 Gene in Radio-Sensitivity of Esophageal Cancer Cell Lines to Irradiation

Background: Radio therapy plays an important role in controlling tumor growth in esophageal cancer patients. Objectives: Our study provided comprehensive information about radio-sensitivity of Hdm2 gene in esophageal cancer cells in cancer cells. Methods: The expression of Hdm2 gene in radiosensitive and radio-resistant esophageal squamous cell carcinoma TE1, TE8 and TE11 cell lines is studied. RNA extracted from each esophageal cancer cell, then Quantitative reverse transcription PCR (RT-qPCR) assay were used for measuring HDM2 gene expression. Finally, radiosensitivity was determined for every three esophageal cancer cell lines. Results: It was found that Hdm2 gene significantly expressed in TE1, TE8 and TE11 cell lines by qRT-PCR analysis. Clonogenic assay showed that TE-11 was very sensitive to radiation relative to other cell lines. Conclusions: Hdm2 protein binding to the P53 protein is a key regulator of cell cycle control and ‎genome protection Cell cycle control, programmed cell death and response cells to ‎radition play important roles.‎

Checkpoint-dependent phosphorylation of Med1/TRAP220 in response to DNA damage.

Mediator complex subunit 1 (Med1)/Thyroid hormone receptor-associated protein 220 (TRAP220), an essential component of thyroid hormone receptor-associated proteins (TRAP)/mediator, plays important roles in hormone responses and tumorigenesis. However, the role of Med1 in the DNA damage response has not been studied. In this study, we found that DNA damage, resulted from γ-irradiation, ultraviolet (UV)-irradiation, or hydroxyurea, induced phosphorylation of Med1 in vivo. Phosphorylation of Med1 was abrogated by either caffeine or wortmannin treatment, suggesting that Med1 is phosphorylated through the DNA damage checkpoint pathway. A checkpoint kinase 1 (Chk1)/checkpoint kinase 2 (Chk2) consensus phosphorylation motif was identified at Serine 671 of Med1 and Ser671 motif was primarily phosphorylated by Chk2 in vitro. Moreover, the in vivo phosphorylation of Med1 was abrogated by a Chk2 inhibitor, and physical interaction between Chk2 and Med1 was observed, confirming that Chk2 is responsible for Med1 phosphorylation upon DNA damage. These results suggest that Med1 is a novel target for the DNA damage checkpoint pathway and may participate in the DNA damage response. Consistent with this notion, knockdown of Med1 expression caused a significant increase in cellular sensitivity toUV irradiation. Moreover, microarray analysis revealed that the UV-induced activation of the transcription of important regulators of cell cycle control and DNA repair, including p21, Gadd45, Rad50, DnaJ, and RecQL, was impaired upon Med1 knockdown. Taken together, our data suggest that Med1 is a novel target for Chk2-mediated phosphorylation and may play a role in cellular DNA damage responses by mediating proper induction of gene transcription upon DNA damage.

Abstract 3565: Dinaciclib, a CDK inhibitor promotes proteasomal degradation of Mcl-1 and enhances ABT-737 mediated cell death in malignant human glioma cell lines

Abstract The prognosis of malignant glioma, the most common brain tumor, is still poor, thus underscoring the need to develop novel treatment strategies. Because glioma cells commonly exhibit genomic alterations involving genes that regulate cell cycle control, there is a strong rationale to examine the potential efficacy of strategies to counteract this process. In this study, we examined the antiproliferative effects of the cyclin-dependent kinase inhibitor dinaciclib in malignant human glioma cell lines, with intact or deleted or mutated p53 or PTEN or p14ARF, or EGFR amplification status. Dinaciclib was found to inhibit cell proliferation and induce cell cycle arrest at the G2/M checkpoint, independent of p53 mutational status. In a standard 72 h MTS assay, at clinically relevant concentrations, dose-dependent antiproliferative effects were observed but cell death was not induced. Moreover, the combination of conventional chemotherapeutic agents and various growth signaling inhibitors with dinaciclib did not yield synergistic cytotoxicity. In contrast, combination of the Bcl-2/Bcl-xL inhibitor ABT-263 or ABT-737 with dinaciclib potentiated the apoptotic response induced by each single drug. The synergistic killing by ABT-737 with dinaciclib led to cell death accompanied by the hallmarks of apoptosis, including an early loss of the mitochondrial transmembrane potential, the release of cytochrome c, smac/DIABLO and apoptosis-inducing factor (AIF), phosphatidylserine exposure on the plasma membrane surface and activation of caspases and PARP. Mechanistic studies revealed that dinaciclib promoted proteasomal degradation of Mcl-1. Citation Format: Esther P. Jane, Daniel Premkumar, Jonathon Cavaleri, Thatchana Rajasekar, Philip Sutera, Ian Pollack. Dinaciclib, a CDK inhibitor promotes proteasomal degradation of Mcl-1 and enhances ABT-737 mediated cell death in malignant human glioma cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3565.

The Oncogenic Roles of DICER1 RNase IIIb Domain Mutations in Ovarian Sertoli-Leydig Cell Tumors

DICER1, an endoribonuclease required for microRNA (miRNA) biogenesis, is essential for embryogenesis and the development of many organs including ovaries. We have recently identified somatic hotspot mutations in RNase IIIb domain of DICER1 in half of ovarian Sertoli-Leydig cell tumors, a rare class of sex-cord stromal cell tumors in young women. These hotspot mutations lost IIIb cleavage activity of DICER1 in vitro and failed to produce 5p-derived miRNAs in mouse Dicer1-null ES cells. However, the oncogenic potential of these hotspot DICER1 mutations has not been studied. Here, we further revealed that the global expression of 5p-derived miRNAs was dramatically reduced in ovarian Sertoli-Leydig cell tumors carrying DICER1 hotspot mutations compared with those without DICER1 hotspot mutation. The miRNA production defect was associated with the deregulation of genes controlling cell proliferation and the cell fate. Using an immortalized human granulosa cell line, SVOG3e, we determined that the D1709N-DICER1 hotspot mutation failed to produce 5p-derived miRNAs, deregulated the expression of several genes that control gonadal differentiation and cell proliferation, and promoted cell growth. Re-expression of let-7 significantly inhibited the growth of D1709N-DICER1 SVOG3e cells, accompanied by the suppression of key regulators of cell cycle control and ovarian gonad differentiation. Taken together, our data revealed that DICER1 hotspot mutations cause systemic loss of 5p-miRNAs that can both drive pseudodifferentiation of testicular elements and cause oncogenic transformation in the ovary.

Effect of human papillomavirus infection on the immune system and its role in the course of cervical cancer.

Human papillomavirus (HPV) is widely known as a cause of cervical intraepithelial neoplasia (CIN) and cervical cancer. The mechanisms involved have been studied by numerous studies. The integration of the virus genome into the host cells results in the abnormal regulation of cell cycle control. HPV can also induce immune evasion of the infected cells, which enable the virus to be undetectable for long periods of time. The induction of immunotolerance of the host's immune system by the persistent infection of HPV is one of the most important mechanisms for cervical lesions. The present review elaborates on the roles of several types of immune cells, such as macrophages and natural killer cells, which are classified as innate immune cells, and dendritic cells (DCs), cluster of differentiation (CD)4+/CD8+ T cells and regulatory T cells, which are classified as adaptive immune cells. HPV infection could effect the differentiation of these immune cells in a unique way, resulting in the host's immune tolerance to the infection. The immune system modifications induced by HPV infection include tumor-associated macrophage differentiation, a compromised cellular immune response, an abnormal imbalance between type 1 T-helper cells (Th1) and Th2 cells, regulatory T cell infiltration, and downregulated DC activation and maturation. To date, numerous types of preventative vaccines have been created to slow down carcinogenesis. Immune response activation-based therapeutic vaccine is becoming more and more attractive for the treatment of HPV-associated diseases.

The NEDD8 inhibitor MLN4924 increases the size of the nucleolus and activates p53 through the ribosomal-Mdm2 pathway

The ubiquitin-like molecule NEDD8 is essential for viability, growth and development, and is a potential target for therapeutic intervention. We found that the small molecule inhibitor of NEDDylation, MLN4924, alters the morphology and increases the surface size of the nucleolus in human and germline cells of Caenorhabditis elegans in the absence of nucleolar fragmentation. SILAC proteomics and monitoring of rRNA production, processing and ribosome profiling shows that MLN4924 changes the composition of the nucleolar proteome but does not inhibit RNA Pol I transcription. Further analysis demonstrates that MLN4924 activates the p53 tumour suppressor through the RPL11/RPL5-Mdm2 pathway, with characteristics of nucleolar stress. The study identifies the nucleolus as a target of inhibitors of NEDDylation and provides a mechanism for p53 activation upon NEDD8 inhibition. It also indicates that targeting the nucleolar proteome without affecting nucleolar transcription initiates the required signalling events for the control of cell cycle regulators.

Perfluorooctanoic acid (PFOA) affects distinct molecular signalling pathways in human primary hepatocytes.

Perfluorooctanoic acid (PFOA) was shown to damage the liver of rodents and to impair embryonic development. At the molecular level, the hepatotoxic effects were attributed to the PFOA-mediated activation of peroxisome proliferator-activated receptor alpha (PPARα). In general, PPARα-dependent effects are less pronounced in humans than in rodents, and the hazard potential of PFOA for humans is controversially discussed. To analyse the effects of PFOA in human hepatocytes, a microarray analysis was conducted to screen for PFOA-mediated alterations in the transcriptome of human primary hepatocytes. A subsequent network analysis revealed that PFOA had an impact on several signalling pathways in addition to the well-known activation of PPARα. The microarray data confirmed earlier findings that PFOA: (i) affects the estrogen receptor ERα, (ii) activates the peroxisome proliferator-activated receptor gamma (PPARγ), and (iii) inhibits the function of the hepatocyte nuclear factor 4α (HNF4α) which is an essential factor for liver development and embryogenesis. Finally, as a novel finding, PFOA was shown to stimulate gene expression of the proto-oncogenes c-Jun and c-Fos. This was confirmed by using the HepG2 cell line as a model for human hepatocytes. PFOA stimulated cellular proliferation and the metabolic activity of the cells, and upregulated the expression of various cyclins which have a central function in the regulation of cell cycle control. Functional studies, however, indicated that PFOA had no impact on c-Jun and c-Fos phosphorylation and on AP-1-dependent gene transcription, thus demonstrating that PFOA-induced proliferation occurs largely independent of c-Jun and c-Fos.

The effects of the fungicides fenhexamid and myclobutanil on SH-SY5Y and U-251 MG human cell lines

Mixtures of pesticides in foodstuffs and the environment are ubiquitous in the developed world and although agents are usually exhaustively tested individually, the toxicological implications of pesticide mixtures are underreported. In this study, the effects of two fungicides, fenhexamid and myclobutanil were investigated individually and in combination on two human cell lines, SH-SY5Y neuronal cells and U-251 MG glial cells. After 48h of incubation with increasing concentrations of pesticides ranging from 1 to 1000μM, gene expression profiles were studied in addition to toxicity end points, including cell viability, mitochondrial depolarisation as well as cellular glutathione maintenance. There were no significant differences between the susceptibility of the two cell lines in terms of cell viability assessment or mitochondrial membrane potential, when agents were administered either individually or in combination. By contrast, in the presence of the fungicides, the SH-SY5Y cells showed significantly greater susceptibility to oxidative stress in terms of total thiol depletion in comparison with the astrocytic cells. Treatment with the two pesticides led to significant changes in the cell lines’ expression of several genes which regulate cell cycle control and growth (RB1, TIMP1) as well as responses to DNA attrition (ATM and CDA25A) and control of apoptosis (FAS). There was no evidence in this study that the combination of fenhexamid and myclobutanil was significantly more toxic than individual exposure, although gene expression changes suggested there may be differences in the sub-lethal response of both cell lines to both individual and combined exposure.

Expression and prognostic role of SGTA in human breast carcinoma correlates with tumor cell proliferation.

Small glutamine-rich tetratricopeptide repeat-containing protein alpha (SGTA) was reported to be implicated in various cellular processes and involved in control of cell cycle regulation and transcription. It may play a critical role in oncogenesis. In this study, to investigate the potential roles of SGTA in breast cancer, expression patterns, interaction and the correlation with clinical/prognostic factors of SGTA and Ki-67 were examined among patients with breast cancer. Immunohistochemistry and Western blot analysis were performed for SGTA in 100 breast carcinoma samples. The data were correlated with clinicopathological features. The univariate and multivariate survival analyses were also performed to determine the prognostic significance. We found that SGTA was overexpressed in breast carcinoma compared with the adjacent normal tissues. High expression of SGTA was positively associated with histological grade (P=0.002) and Ki-67 (P=0.001). Univariate analysis showed that SGTA expression was associated with a poor prognosis (P=0.002). Kaplan-Meier survival curves of the study population showed that high expression level of SGTA significantly correlated with short-term survival. While in vitro, SGTA depletion by small interfering RNA inhibited cell proliferation and cell cycle in breast cancer cell lines. Western blot analyses showed that SGTA depletion decreased cyclin A, cyclin B and CDK2, whereas increased p27 levels. Additionally, treatment of phosphatidylinositol 3-kinase inhibitor LY294002 could arrest cells growth and diminish SGTA expression. These results suggested that SGTA overexpression was involved in the pathogenesis of breast cancer which might serve as a future target for novel treatment in breast cancer.

The Knockout of Rap1 Accelerates Liver Regeneration After Transplantation.

Introduction and aim: Impaired of liver regeneration severe affects the tissue repair following liver surgery. Cytokine, growth factor and metabolic networks play important roles in theprogression of liver regeneration. Rap1 is a part of the shelterin complex at mammalian telomeres, involved in protecting chromosome ends and promoting gene silencing. Recent research revealed its novel roles in the regulation of metabolism andinflammatory response through binding to extra-telomeric sites. We recently also showed that the knockout of Rap1 attenuated liver graft injury after transplantation. Here, we aimed to investigate the role of Rap1 in liver regeneration and to explore underlying mechanism. Methods:Clinically, the intragraft Rap1 expression and its correlation among liver function, inflammatory cytokines/chemokines, and macrophage infiltration were analyzed in human liver graft biopsies after transplantation. To investigate the direct role of Rap1 in hepatocyte proliferation and liver regeneration, Rap1 knockoutand wild type mice were subjected to major hepatectomy plus partial hepatic ischemia/reperfusion injury (IRI). Mouse hepatocyte proliferation, histological damage, liver function and gene expressions were compared between Rap1 knockoutand wild type group. Results: Intrahepatic Rap1 expression was increased in small-for-size graft in comparison to normal graft (2.9 vs 1.9 folds of normal liver, p<0.05), and associated with higher expressions of inflammatory cytokines/chemokines and more infiltrations of macrophage and neutrophil at 2 hours after transplantation. Furthermore, overexpression of Rap1 was significantly correlated with impaired liver function after transplantation (ALT: day 1, 3, 4 p<0.05; AST: day 0, 3, 4, 5 p<0.05). In mouse model, the knockout of Rap1 significantly accelerated liver regeneration at day 2 after major hepatectomy and hepatic IRI compared to wild type group (Ki67: 56 vs 11/HPF, p<0.05; PCNA: 46 vs 29/HPF, p<0.05). The knockout of Rap1 also attenuated histological damage and liver function. Furthermore, reduced expression of PPAR-α and increased expressions of G0S2 and IL1β were detected in Rap1 knockout mice. Conclusion: The knockout of Rap1 accelerates liver regeneration and associated with the regulation of cell cycle control, PPAR-α and cytokine signaling.

Immunologic treatments for precancerous lesions and uterine cervical cancer

Development of HPV-associated cancers not only depends on efficient negative regulation of cell cycle control that supports the accumulation of genetic damage, but also relies on immune evasion that enable the virus to go undetected for long periods of time. In this way, HPV-related tumors usually present MHC class I down-regulation, impaired antigen-processing ability, avoidance of T-cell mediated killing, increased immunosuppression due to Treg infiltration and secrete immunosuppressive cytokines. Thus, these are the main obstacles that immunotherapy has to face in the treatment of HPV-related pathologies where a number of different strategies have been developed to overcome them including new adjuvants. Although antigen-specific immunotherapy induced by therapeutic HPV vaccines was proved extremely efficacious in pre-clinical models, its progression through clinical trials suffered poor responses in the initial trials. Later attempts seem to have been more promising, particularly against the well-defined precursors of cervical, anal or vulvar cancer, where the local immunosuppressive milieu is less active. This review focuses on the advances made in these fields, highlighting several new technologies (such as mRNA vaccine, plant-derived vaccine). The most promising immunotherapies used in clinical trials are also summarized, along with integrated strategies, particularly promising in controlling tumor metastasis and in eliminating cancer cells altogether.After the early promising clinical results, the development of therapeutic HPV vaccines need to be implemented and applied to the users in order to eradicate HPV-associated malignancies, eradicating existing perception (after the effectiveness of commercial preventive vaccines) that we have already solved the problem.

A review of epigenetic and gene expression alterations associated with intracranial meningiomas

A more comprehensive understanding of the epigenetic abnormalities associated with meningioma tumorigenesis, growth, and invasion may provide useful targets for molecular classification and development of targeted therapies for meningiomas. The authors performed a review of the current literature to identify the epigenetic modifications associated with the formation and/or progression of meningiomas. Several epigenomic alterations, mainly pertaining to DNA methylation, have been associated with meningiomas. Hypermethylation of TIMP3 inactivates its tumor suppression activity while CDKN2 (p14[ARF]) and TP73 gene hypermethylation and HIST1H1c upregulation interact with the p53 regulation of cell cycle control. Other factors such as HOX, IGF, WNK2, and TGF-β epigenetic modifications allow either upregulation or downregulation of critical pathways for meningioma development, progression, and recurrence. Genome-wide methylation profiling demonstrated that global hypomethylation correlates with tumor grades and severity. Identification of additional epigenetic changes, such as histone modification and higher-order chromosomal structure, may allow for a more thorough understanding of tumorigenesis and enable future individualized treatment strategies for meningiomas.

Abstract 1333: Specific RB1 mutations and risk of subsequent neoplasms among survivors of hereditary retinoblastoma.

Abstract The retinoblastoma tumor suppressor gene (RB1) is a key regulator of cell cycle control, most notably through E2F transcription factor binding. Deregulation of RB1 is important in many cancers. Survivors of hereditary retinoblastoma, caused by a germline mutation in RB1, experience substantially elevated risks for subsequent neoplasms, particularly bone and soft tissue sarcomas and melanomas. Oncogenic RB1 mutations arise from a range of DNA alterations and occur throughout the gene. No previous study has evaluated how the specific location and functional consequences of these RB1 mutations may differentially confer risk for specific subsequent neoplasms. As part of a biomarker study nested within a long-term follow-up study of the risk of subsequent cancers in retinoblastoma survivors, we identified germline RB1 mutations in 76/80 (95%) survivors of hereditary retinoblastoma who developed at least one subsequent neoplasm and had sufficient DNA for mutation testing (median follow-up time=47 years, range 7-71 years). Survivors were classified into four groups according to whether they ever developed a soft tissue sarcoma (STS, N=47), melanoma (without STS, N=10), bone tumor (without STS, N=5), or another type of subsequent malignant neoplasm (N=14). The identified mutations were distributed throughout RB1, from the promoter through exon 25. Nonsense, frameshift, or splice mutations that resulted in premature termination of the RB1 residue sequence were identified in 51 (67%) survivors, with the remaining classified as having loss of multiple exons (N=6), loss or rearrangement of a single exon (N=5), a splice mutation (N=7), a missense mutation (N=4), or an intronic substitution with unknown consequence (N=3). Preliminary analyses indicated that RB1 mutations resulting in loss of &amp;gt;2 exons were more common in survivors with a subsequent STS (40/47, 85%) than those with a subsequent melanoma (6/10, 60%) or bone tumor (2/5, 40%; Fisher's exact P&amp;lt;0.001). In an analysis of the RB1 regions impacted by the mutation, those that affected E2F binding (including mutations affecting the E2F binding site, Domain B, or Domain C) were more common in survivors with a subsequent STS (46/47, 98%) or melanoma (9/10, 90%) than those with a bone tumor (2/5, 40%; Fisher's exact P=0.020). Further analyses will include 14 additional survivors who developed a subsequent neoplasm for whom mutation testing is ongoing, and comparison of the spectrum of RB1 mutations in these survivors to hereditary retinoblastoma survivors without a subsequent neoplasm. If confirmed, such correlations between specific RB1 mutations and subsequent neoplasm risk may inform long-term surveillance practices for hereditary retinoblastoma survivors and provide insight into the key role of RB1 in oncogenesis. Citation Format: Lindsay M. Morton, Jeannette R. Wong, Brenda L. Gallie, David H. Abramson, Johanna M. Seddon, Michael Dean, Bert Gold, Alisa M. Goldstein, Ruth A. Kleinerman, Margaret A. Tucker. Specific RB1 mutations and risk of subsequent neoplasms among survivors of hereditary retinoblastoma. [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 1333. doi:10.1158/1538-7445.AM2013-1333

Integrated Analysis of Whole-Exome Sequencing and Micrornas Expression in Blast Crisis Transformation of Chronic Myeloid Leukemia

AbstractAbstract 3727 Objective.The molecular events leading to the evolution of BCR/ABL+ chronic myeloid leukemia (CML) from the chronic phase (CP) to the advanced phase (blast crisis-BP) are poorly understood. The aggressiveness of BP and the poor over-all survival of BP patients needs deep investigation of the biological basis of blastic transformation. We here present the results obtained from the analysis of paired (CP)/(BP) CML samples from patients that underwent progression after standard therapy. The access to matched CP/BP samples will render this data highly valuable. Methods.We performed whole-exome sequencing analysis using high-throughput technologies (Illumina Genome Analyzer IIx) from genomic DNA of four paired samples. The cross-match between BP and CP exomes was performed with dedicated in-house C#software. Gross chromosomal rearrangements were evaluated using CEQer(Comparative-Exonic-Quantification-Analyzer) software from whole-exome sequencing data. We also evaluated microRNA(miRNA) differential expression extracting RNA from five paired samples, using Nanostring nCounter miRNA expression assay. Differentially-expressed miRNAs with a p-value<0.001 were considered significant. Putative targets of significantly deregulated miRNAs were generated using miRGen software analysis(http://www.diana.pcbi.upenn.edu/miRGen.html). Results.By comparing exome-sequences of four paired CP (used as a control) and BP samples we found a total of 8 single nucleotide somatic mutations. Among the 8 variants identified, 4 of them ranked >1 in the GenRanker cancer scoring system (http://cbio.mskcc.org/tcga-generanker). We show here that, unexpectedly, blast crisis samples have a limited number of acquired mutations compared to chronic phases (average=2 mutations/patient) with patient number two and four displaying the lowest and the higher frequencies, respectively (patient n.2=0 mutations, patient n.4=4 mutations). CEQer analysis of whole exome data showed that 3/4 patients present gross chromosomal rearrangements of at least 2 chromosomes (bulky alteration of chromosome7 present in 2/4 patients); critical regulators of cell cycle control (e.g. CDKN2A and p53) have also been shown to be deleted in patient number 1 and 4, respectively. The individual mutations and rearrangements identified will be presented at the meeting.Differential expression of miRNAs showed that miR-106a, miR-17, miR-20a and miR-20b were significantly down-regulated while miR-148a was significantly up-regulated in all the blast crisis compared to chronic phase samples. In-silico analysis of the putative deregulated targets revealed a strong enrichment of genes involved in molecular mechanisms of cancer, with the 20% of these genes involved in cell cycle regulation. The integrated analysis of these informative data will help to understand the molecular mechanisms responsible for blast crisis progression. Disclosures:No relevant conflicts of interest to declare.