Accumulation Of p27Kip1 Research Articles

Prolonged cetuximab treatment promotes p27Kip1-mediated G1 arrest and autophagy in head and neck squamous cell carcinoma

Cetuximab, an anti-epidermal growth factor receptor (EGFR) monoclonal antibody, is an efficient anti-tumor therapeutic agent that inhibits the activation of EGFR; however, data related to the cellular effects of prolonged cetuximab treatment are limited. In this study, the long-term cellular outcome of prolonged cetuximab treatment and the related molecular mechanism were explored in a head and neck squamous cell carcinoma cell line constitutively expressing a fluorescent ubiquitination-based cell cycle indicator. Fluorescent time-lapse imaging was used to assess clonal growth, cell motility, and cell-cycle progression. Western blot analysis was performed to measure the level of phosphorylation and protein-expression following cetuximab treatment. Over 5 days cetuximab treatment decreased cell motility and enhanced G1 phase cell arrest in the central region of the colonies. Significantly decreased phosphorylation of retinoblastoma, Skp2, and Akt-mTOR proteins, accumulation of p27Kip1, and induction of type II LC3B were observed over 8 days cetuximab treatment. Results of the present study elucidate the cetuximab-dependent inhibition of cell migration, resulting in high cell density-related stress and persistent cell-cycle arrest at G1 phase culminating in autophagy. These findings provide novel molecular insights related to the anti-tumor effects of prolonged cetuximab treatment with the potential to improve future therapeutic strategy.

The Capsid Protein VP1 of Coxsackievirus B Induces Cell Cycle Arrest by Up-Regulating Heat Shock Protein 70.

Manipulating cell cycle is one of the common strategies used by viruses to generate favorable cellular environment to facilitate viral replication. Coxsackievirus B (CVB) is one of the major viral pathogens of human myocarditis and cardiomyopathy. Because of its small genome, CVB depends on cellular machineries for productive replication. However, how the structural and non-structural components of CVB would manipulate cell cycle is not clearly understood. In this study, we demonstrated that the capsid protein VP1 of CVB type 3 (CVB3) induced cell cycle arrest at G1 phase. G1 arrest was the result of the decrease level of cyclin E and the accumulation of p27Kip1. Study on the gene expression profile of the cells expressing VP1 showed that the expression of both heat shock protein 70-1 (Hsp70-1) and Hsp70-2 was significantly up-regulated. Knockdown of Hsp70 resulted in the increased level of cyclin E and the reduction of p27Kip1. We further demonstrated that the phosphorylation of the heat shock factor 1, which directly promotes the expression of Hsp70, was also increased in the cell expressing VP1. Moreover, we show that CVB3 infection also induced G1 arrest, likely due to dysregulating Hsp70, cyclin E, and p27, while knockdown of Hsp70 dramatically inhibited viral replication. Cell cycle arrest at G1 phase facilitated CVB3 infection, since viral replication in the cells synchronized at G1 phase dramatically increased. Taken together, this study demonstrates that the VP1 of CVB3 induces cell cycle arrest at G1 phase through up-regulating Hsp70. Our findings suggest that the capsid protein VP1 of CVB is capable of manipulating cellular activities during viral infection.

Chronic p27Kip1 Induction by Dexamethasone Causes Senescence Phenotype and Permanent Cell Cycle Blockade in Lung Adenocarcinoma Cells Over-expressing Glucocorticoid Receptor

Dexamethasone (Dex), co-administered to lung adenocarcinoma patients with pemetrexed chemotherapy, protects against pemetrexed cytotoxicity by inducing reversible G1 arrest, reflected by the effect of Dex on FLT-PET images of patient tumors. However, perioperative Dex treatment increases survival but the mechanism is unknown. In cells with glucocorticoid receptor-α (GR) expression corresponding to higher clinical tumor levels, Dex-induced growth arrest was followed by marked cell expansion, beta-galactosidase expression and Ki67 negativity, despite variable p53 and K-RAS status. Dex induced a transient early surge in p21Cip1. However, a progressive, irreversible loss of clonogenic growth, whose time of onset was dependent on GR level and Dex dose, was independent of p21Cip1and caused by gradual accumulation of p27Kip1 due to transcriptional activation of p27Kip1 by Dex. This effect was independent of canonical pathways of senescence or p27Kip1 regulation. The in vitro observations were reflected by growth suppression and P27Kip1 induction in GR-overexpressing tumor xenografts compared with isogenic low-GR tumors. Extended Dex treatment induces irreversible cell cycle blockade and a senescence phenotype through chronic activation of the p27Kip1 gene in GR overexpressing lung tumor cell populations and hence could improve outcome of surgery/pemetrexed chemotherapy and sensitize tumors to immunotherapy.

Antitumor Effects of Blocking Protein Neddylation in T315I-BCR-ABL Leukemia Cells and Leukemia Stem Cells.

Imatinib revolutionized the treatment of chronic myeloid leukemia (CML), but drug resistance and disease recurrence remain a challenge. In this study, we suggest a novel strategy based on blocking protein neddylation to address BCR-ABL point mutations and leukemia stem cells (LSC) that lie at the root of imatinib-resistant recurrences. On the basis of the finding that the NEDD8-activating enzyme subunit NAE1 is overexpressed in CML cells, we hypothesized that the function of certain neddylation-dependent protein substrates might be targeted to therapeutic ends in imatinib-resistant CML cells and LSCs. In support of this hypothesis, we demonstrated that the NAE1 inhibitor MLN4924 induced G2-M-phase arrest and apoptosis in bulk CML cells with wild-type p53, regardless of their T315I mutation status in BCR-ABL. Moreover, MLN4924 inhibited the survival and self-renewal of primary human CML CD34+ cells and LSCs in CML-bearing mice via accumulation of p27kip1 in the nucleus. Notably, p27kip1 silencing attenuated the suppressive effect of MLN4924 on the maintenance of LSCs in CML-bearing mice. Taken together, our findings offer a preclinical proof of concept for targeting protein neddylation as a novel therapeutic strategy to override mutational and LSC-derived imatinib resistance in CML.Significance: These findings highlight a mediator of protein neddylation, a type of protein turnover mechanism, as a viable therapeutic target against imatinib-resistant forms of chronic myelogenous leukemia. Cancer Res; 78(6); 1522-36. ©2018 AACR.

Asxl1 deficiency in embryonic fibroblasts leads to cellular senescence via impairment of the AKT-E2F pathway and Ezh2 inactivation

Although ASXL1 mutations are frequently found in human diseases, including myeloid leukemia, the cell proliferation–associated function of ASXL1 is largely unknown. Here, we explored the molecular mechanism underlying the growth defect found in Asxl1-deficient mouse embryonic fibroblasts (MEFs). We found that Asxl1, through amino acids 371 to 655, interacts with the kinase domain of AKT1. In Asxl1-null MEFs, IGF-1 was unable to induce AKT1 phosphorylation and activation; p27Kip1, which forms a ternary complex with ASXL1 and AKT1, therefore remained unphosphorylated. Hypophosphorylated p27Kip1 is able to enter the nucleus, where it prevents the phosphorylation of Rb; this ultimately leads to the down-regulation of E2F target genes as confirmed by microarray analysis. We also found that senescence-associated (SA) genes were upregulated and that SA β-gal staining was increased in Asxl1−/− MEFs. Further, the treatment of an AKT inhibitor not only stimulated nuclear accumulation of p27Kip1 leading to E2F inactivation, but also promoted senescence. Finally, Asxl1 disruption augmented the expression of p16Ink4a as result of the defect in Asxl1-Ezh2 cooperation. Overall, our study provides the first evidence that Asxl1 both activates the AKT-E2F pathway and cooperates with Ezh2 through direct interactions at early embryonic stages, reflecting that Asxl1 disruption causes cellular senescence.

Inhibiting the role of Skp2 suppresses cell proliferation and tumorigenesis of human gastric cancer cells via the upregulation of p27kip1.

Gastric cancer is a malignant disease of the digestive system with high rates of incidence and mortality. S‑phase kinase‑associated protein 2 (Skp2) is a novel oncogene, which has been identified to be important in tumor progression and metastasis. In order to clarify the role of Skp2 in human gastric cancer, the present study detected the expression of Skp2 in human gastric cancer tissues, and investigated the molecular mechanism of Skp2 in the progression of gastric carcinoma. The results of the initial bioinformatics analysis showed that Skp2 was significantly upregulated in 31specimens of primary gastric cancer from a UK patient cohort, and in 10 gastric cancer lines of a side population, compared with normal gastric tissues (P<0.01). Specimens from 47patients with gastric cancer and 19 normal gastric tissue specimens were obtained and analyzed using western blot analysis. The positive rate of expression of Skp2 was 87.2%, indicating that the expression of Skp2 was observed in 41specimens of the detected gastric cancer samples, whereas the positive rate of the expression of Skp2 was 5.6% in the normal gastric samples (P<0.01). In the human gastric cancer cell lines, the defective regulation of Skp2 or presence of an Skp2 inhibitor inhibited the proliferation of BGC‑823 and MKN‑45 cells. In addition, the Skp2 inhibitor suppressed the proliferation of gastric cancer cells in a time‑ and dose‑dependent manner. Furthermore, transfection with Skp2 short hairpin (sh)RNA or treatment with SKPinhibitorC1 for 48 and 72h led to the accumulation of p27kip1 in Hela cells. Tumorigenicity experiments involving nude mice showed that interference of the expression of Skp2 inhibited the growth of the human gastric tumor cells in the nude mice, and the tumor weights and volumes in the Skp2 shRNA group were significantly lower, compared with those in the negative control shRNA group (P<0.01) and untreated group (P<0.01). Taken together, these data suggested that Skp2 acted as an oncogene in human gastric cancer, and that Skp2‑mediated p27kip1 degradation contributed to the progression of gastric cancer. Abrogating the effects of Skp2 may effectively inhibit the growth of gastric cancer cells, which may be useful as a novel target in the clinical treatment of gastric cancer.

Combined inhibition of EZH2 and histone deacetylases as a potential epigenetic therapy for non-small-cell lung cancer cells.

Recent discoveries have revealed that human cancer involves aberrant epigenetic alterations. We and others have previously shown that the histone methyltransferase EZH2, the catalytic subunit of polycomb repressive complex 2 (PRC2), is frequently overexpressed in non‐small‐cell lung cancer (NSCLC) and that an EZH2 inhibitor, 3‐deazaneplanocin A, inhibits the proliferation of NSCLC cells. Transcriptional silencing by EZH2 was recently shown to be required for the activity of histone deacetylases (HDACs) that interact with another PRC2 protein, EED. To develop a more effective epigenetic therapy for NSCLC, we determined the effects of co‐treatment with 3‐deazaneplanocin A and the HDAC inhibitor vorinostat (SAHA) in NSCLC cells. The co‐treatment synergistically suppressed the proliferation of all tested NSCLC cell lines, regardless of their epidermal growth factor receptor (EGFR) status. The synergistic effect was associated with slightly decreased histone H3 lysine 27 trimethylation, modestly increased histone acetylation, and the depletion of EZH2 and other PRC2 proteins. The co‐treatment resulted in an accumulation of p27Kip1, decrease in cyclin A, and increased apoptotic fraction in an additive/synergistic manner. Interestingly, the co‐treatment strongly suppressed EGFR signaling, not only in EGFR‐wild‐type NSCLC cells, but also in EGFR‐mutant cells, mainly through dephosphorylation of EGFR. Furthermore, the co‐treatment suppressed the in vivo tumor growth of EGFR‐mutant, EGFR–tyrosine kinase‐resistant H1975 cells more effectively than did each agent alone, without visible toxicity. These results suggest that the combined pharmacological targeting of EZH2 and HDACs may provide more effective epigenetic therapeutics for NSCLC.

Bortezomib-mediated downregulation of S-phase kinase protein-2 (SKP2) causes apoptotic cell death in chronic myelogenous leukemia cells.

BackgroundProteasome inhibitors are attractive cancer therapeutic agents because they can regulate apoptosis-related proteins. Bortezomib also known as Velcade®, a proteasome inhibitor that has been approved by the food and drug administration for treatment of patients with multiple myeloma, and many clinical trials are ongoing to examine to the efficacy of bortezomib for the treatment of other malignancies. Bortezomib has been shown to induce apoptosis and inhibit cell growth of many cancer cells. In current study, we determine whether bortezomib induces cell death/apoptosis in CML.MethodsCell viability was measured using MTT assays. Apoptosis was measured by annexin V/PI dual staining and DNA fragmentation assays. Immunoblotting was performed to examine the expression of proteins. Colony assays were performed using methylcellulose.ResultsTreatment of CML cells with bortezomib results in downregulation of S-phase kinase protein 2 (SKP2) and concomitant stabilization of the expression of p27Kip1. Furthermore, knockdown of SKP2 with small interference RNA specific for SKP2 caused accumulation of p27Kip1. CML cells exposed to bortezomib leads to conformational changes in Bax protein, resulting in loss of mitochondrial membrane potential and leakage of cytochrome c to the cytosol. In the cytosol, cytochrome c causes sequential activation of caspase-9, caspase-3, PARP cleavage and apoptosis. Pretreatment of CML cells with a universal inhibitor of caspases, z-VAD-fmk, prevents bortezomib-mediated apoptosis. Our data also demonstrated that bortezomib treatment of CML downregulates the expression of inhibitor of apoptosis proteins. Finally, inhibition of proteasome pathways by bortezomib suppresses colony formation ability of CML cells.ConclusionsAltogether, these findings suggest that bortezomib suppresses the cell proliferation via induction of apoptosis in CML cells by downregulation of SKP2 with concomitant accumulation of p27Kip1, suggesting that proteasomal pathway may form novel therapeutic targets for better management of CML.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-016-0823-y) contains supplementary material, which is available to authorized users.

Pioglitazone, a PPARγ agonist, attenuates PDGF-induced vascular smooth muscle cell proliferation through AMPK-dependent and AMPK-independent inhibition of mTOR/p70S6K and ERK signaling

Pioglitazone (PIO), a PPARγ agonist that improves glycemic control in type 2 diabetes through its insulin-sensitizing action, has been shown to exhibit beneficial effects in the vessel wall. For instance, it inhibits vascular smooth muscle cell (VSMC) proliferation, a major event in atherosclerosis and restenosis after angioplasty. Although PPARγ-dependent and PPARγ-independent mechanisms have been attributed to its vasoprotective effects, the signaling events associated with PIO action in VSMCs are not fully understood. To date, the likely intermediary role of AMP-activated protein kinase (AMPK) toward PIO inhibition of VSMC proliferation has not been examined. Using human aortic VSMCs, the present study demonstrates that PIO activates AMPK in a sustained manner thereby contributing in part to inhibition of key proliferative signaling events. In particular, PIO at 30μM concentration activates AMPK to induce raptor phosphorylation, which diminishes PDGF-induced mTOR activity as evidenced by decreased phosphorylation of p70S6K, 4E-BP1, and S6 and increased accumulation of p27kip1, a cell cycle inhibitor. In addition, PIO inhibits the basal phosphorylation of ERK in VSMCs. Downregulation of endogenous AMPK by target-specific siRNA reveals an AMPK-independent effect for PIO inhibition of ERK, which contributes in part to diminutions in cyclin D1 expression and Rb phosphorylation and the suppression of VSMC proliferation. Furthermore, AMPK-dependent inhibition of mTOR/p70S6K and AMPK-independent inhibition of ERK signaling occur regardless of PPARγ expression/activation in VSMCs as evidenced by gene silencing and pharmacological inhibition of PPARγ. Strategies that utilize nanoparticle-mediated PIO delivery at the lesion site may limit restenosis after angioplasty without inducing PPARγ-mediated systemic adverse effects.

PPAR-γ Agonists Induce Growth Arrest of Kit/AML1-ETO Leukemia Cells Acting on Diverse Pathways

Translocation 8;21, t(8;21), with an incidence of up to 12% is among the most common genetic aberrations found in Acute Myeloid leukemias (AMLs). t(8;21) rearranges the AML1 gene on chromosome 21 and the ETO gene on chromosome 8, generating an AML1-ETO fusion protein. It acts as dominant-negative repressor of genes involved in cell cycle control and myeloid differentiation. Despite its critical role in tumor initiation, AML1-ETO is not sufficient, per se, to cause an overt leukemia. It has to cooperate with other genetic alterations, such as those involving receptor tyrosine kinases. KIT tyrosine kinase gain-of-function mutations have been detected in about 50% of t(8;21) AMLs.Peroxisome Proliferator-Activated Receptor-γ (PPAR-γ) agonists are known to inhibit proliferation and induce apoptosis in cancer cells, including those from hematologic malignancies. Despite several promising pre-clinical studies, results of clinical trials are not conclusive so far.We investigated the effects and mechanisms of action of some PPAR-γ agonists in an AML1-ETO/Kit activated leukemia cell line: Kasumi-1. Troglitazone (TGZ) and rosiglitazone (RGZ) reduced proliferation rate and clonogenic potential of Kasumi-1, inducing G1 arrest and apoptosis in a dose and time dependent manner. Activated Kit induces proliferation mostly through the Src kinases/Ras/MAPK pathway. Erk1/2 is constitutively phosphorylated in Kasumi-1 cells and exposure to troglitazone (50μM) significantly reduced its phosphorylated state even if not as much as imatinib. The Kit signaling cascades include activation of PI3-kinase/AKT pathway, but surprisingly TGZ treatment did not affect AKT phosphorylation.It has been shown that PPAR agonists 2-cyano-3,12-dioxo-oleana-1,9(11)-dien-28-oic acid (CDDO) and 15-Deoxy-Δ12,14-ProstaglandinJ2 (15d-PGJ2) are able to suppress TGFβ-induced phosphorylation of AktS473, leading an anti-fibrotic activity in the lung. CDDO and 15d-PGJ2 inhibition effect have been demonstrated direct, PPAR-γindependent and mostly related to electrophilic carbons present in their structures. In the same cellular context rosiglitazone was found relatively poorly effective. We compared, in Kasumi-1 cells, activity of 15d-PGJ2 and CAY10410, two PPAR agonists with similar structure but while 15d-PGJ2 has an electrophilic center CAY10410 does not. CAY10410 is a strong PPAR agonist but was unable to induce any reduction in Kasumi-1 cells proliferation rate or induce apoptosis whereas 15d-PGJ2 strongly inhibited Kasumi-1 growth and induced apoptosis even more efficiently than troglitazone. Molecular characterization of such functional effect is of extreme interest and is ongoing.TGZ and RGZ induced up-regulation of p27Kip1 and p21cip1. We found that it occurs by reduced proteosomal degradation and accumulation of p27kip1 due to up-regulation of Connexin 43 (Cx43) and consequent downregulation of Skp2. AML1-ETO has been reported as Cx43 transcriptional inducer. Upregulation of p21Waf1 seems to be, in preliminary experiments, mostly transcriptional and AML1-ETO direct induction of p21Waf1has been described as well. Interestingly TGZ seems to restore AML1-ETO functions counteracted upon Kit constitutive activation.Some, if not all, the effects might be receptor independent since they were just minimally lessened by a pre-treatment with high dose of GW9662, an irreversible PPAR-γ antagonist that covalently binds to the active site of PPAR-γ.The overall prognosis of AML patients remains severe and AML incidence is expected to increase with the aging population, forcing the need for less toxic regimens in patients with co-morbid conditions precluding intensive chemotherapy. PPAR agonists are promising candidates as specific anticancer drugs within low toxicity multidrug regimens. However, the effectiveness of PPAR ligands seems to vary as various compounds, targeting distinct leukemic cells, behavior differently, because different are the leading molecular mechanism evoked. Their precise clarification will define the real extent of PPAR agonists’ therapeutic potential. DisclosuresNo relevant conflicts of interest to declare.

Abstract 1325: Profilin-1 overexpression downregulates phosphorylation of p27kip1 leading to its nuclear accumulation in breast cancer cells

Abstract Profilin-1 is a ubiquitously expressed actin-binding protein that is downregulated in human breast cancer. Overexpression of profilin-1 in human breast cancer cells suppresses their tumorigenic potential. We had previously shown that profilin-1 overexpression elevates the expression level of p27kip1 leading to cell-cycle arrest in G1 phase. In the present work, we explore the underlying mechanism. Our studies showed that profilin-1-induced accumulation of p27kip1 in breast cancer cells occurs most dramatically in the nuclear compartment and this is due to impaired protein degradation. Consistent with these observations, we found that phosphorylation of p27kip1 at T187 (a prerequisite for p27kip1 degradation by SCF-Skp2 complex in the nucleus) and S10 (a post-translational event that promotes nuclear export of p27kip1) residues are both significantly inhibited by profilin-1 overexpression. Elevating profilin-1 level suppresses AKT activation, and hyper-activating AKT through overexpression of a constitutively active mutant relieves profilin-1-induced inhibition of T187 phosphorylation and elevation of p27kip1. These data demonstrate that profilin-1 can control the cellular level of p27kip1 in breast cancer cells through affecting its post-translational modification in an AKT-dependent pathway. These findings may provide mechanistic insights underlying the tumor-suppressive action of profilin-1. Citation Format: Chang Jiang, William Veon, Partha Roy. Profilin-1 overexpression downregulates phosphorylation of p27kip1 leading to its nuclear accumulation in breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1325. doi:10.1158/1538-7445.AM2014-1325

Abstract 5162: Effects of combined epigenetic therapy with histone methyltransferase EZH2 inhibitor 3-deazaneplanocin and histone deacetylase inhibitor SAHA on non-small cell lung cancer cells

Abstract [Background] EZH2, a polycomb group protein, has histone methyltransferase activity, and is involved in malignant transformation of several cancers. We have previously shown that an EZH2 inhibitor, DZNep, inhibits growth of non-small cell lung cancer(NSCLC) cell lines via G1 arrest and apoptosis. Recently, cotreatment with DZNep and an histone deacetylase (HDAC) inhibitor has been shown to induce apoptosis synergistically in AML, ovarian cancers and renal cancers. Therefore, we determined the combined effect of DZNep and an HDAC inhibitor, SAHA, on NSCLC cells. [Methods] We evaluated the effect of combined therapy with DZNep and SAHA against four human NSCLC cell lines_H1299, H1975, A549 and PC-3. Cell proliferation was measured by MTT assay. Percentage of apoptotic cells was measured using Annexin V-FITC with a flow cytometer. Western blot analysis was performed on total cell lysates. For in vivo assay, H1975 cells were subcutaneously implanted into the flank of BALB/cAJcl nu/nu nude mice. When the average tumor volume reached approximately 50∼100mm3, the following treatments were intraperitoneally administered twice per week for 6 weeks; vehicle alone (5% DM SO), DZNep (4mg/kg), SAHA (40mg/kg), and DZNep (4mg/kg) plus SAHA (40mg/kg). [Results] Co-treatment with DZNep and SAHA inhibited cell proliferation synergistically, and reduced EZH2 expression and histone H3 lysine 27 trimethylation more effectively compared with each agent alone. The co-treatment greatly induced accumulation of p27 Kip1 and decrease in cyclin A expression. Flow cytometry analysis demonstrated that the apoptotic fraction was increased in an additive or synergistic manner by the combination therapy. These effects were more evident in H1975 and PC-3 cells with EGFR mutation, in which the co-treatment markedly reduced phosphorylation of EGFR, AKT and ERK1/2. The combined therapy reduced the levels of β-catenin, and its downstream pro-proliferative targets cyclin D1 and EGFR. The co-treatment with DZNep and SAHA also caused significantly greater inhibition of tumor growth of H1975 xenografts than each agent alone in nude mice without visible toxicity. [Conclusion] Combined epigenetic therapy with an EZH2 inhibitor and an HDAC inhibitor may represent an effective approach for NSCLCs. Citation Format: Taichi Takashina, Ichiro Kinoshita, Junko Kikuchi, Yasushi Shimizu, Jun Sakakibara Konishi, Satoshi Oizumi, Masaharu Nishimura, Hirotoshi Dosaka Akita. Effects of combined epigenetic therapy with histone methyltransferase EZH2 inhibitor 3-deazaneplanocin and histone deacetylase inhibitor SAHA on non-small cell lung cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5162. doi:10.1158/1538-7445.AM2014-5162

Silencing of KIF14 interferes with cell cycle progression and cytokinesis by blocking the p27(Kip1) ubiquitination pathway in hepatocellular carcinoma.

Although it has been suggested that kinesin family member 14 (KIF14) has oncogenic potential in various cancers, including hepatocellular carcinoma (HCC), the molecular mechanism of this potential remains unknown. We aimed to elucidate the role of KIF14 in hepatocarcinogenesis by knocking down KIF14 in HCC cells that overexpressed KIF14. After KIF14 knockdown, changes in tumor cell growth, cell cycle and cytokinesis were examined. We also examined cell cycle regulatory molecules and upstream Skp1/Cul1/F-box (SCF) complex molecules. Knockdown of KIF14 resulted in suppression of cell proliferation and failure of cytokinesis, whereas KIF14 overexpression increased cell proliferation. In KIF14-silenced cells, the levels of cyclins E1, D1 and B1 were profoundly decreased compared with control cells. Of the cyclin-dependent kinase inhibitors, the p27Kip1 protein level specifically increased after KIF14 knockdown. The increase in p27Kip1 was not due to elevation of its mRNA level, but was due to inhibition of the proteasome-dependent degradation pathway. To explore the pathway upstream of this event, we measured the levels of SCF complex molecules, including Skp1, Skp2, Cul1, Roc1 and Cks1. The levels of Skp2 and its cofactor Cks1 decreased in the KIF14 knockdown cells where p27Kip1 accumulated. Overexpression of Skp2 in the KIF14 knockdown cells attenuated the failure of cytokinesis. On the basis of these results, we postulate that KIF14 knockdown downregulates the expression of Skp2 and Cks1, which target p27Kip1 for degradation by the 26S proteasome, leading to accumulation of p27Kip1. The downregulation of Skp2 and Cks1 also resulted in cytokinesis failure, which may inhibit tumor growth. To the best of our knowledge, this is the first report that has identified the molecular target and oncogenic effect of KIF14 in HCC.

Antitumoral Activity of Lenalidomide in In Vitro and In Vivo Models of Mantle Cell Lymphoma Involves the Destabilization of Cyclin D1/p27KIP1 Complexes

Clinical responses to the immmunomodulatory drug lenalidomide have been observed in patients with relapsed/refractory mantle cell lymphoma (MCL), although its mechanism of action remains partially unknown. We investigated whether the expression and subcellular localization of cyclin D1, a major cell-cycle regulator overexpressed in MCL, and the cyclin-dependent kinase inhibitor p27(KIP1), could identify MCL cases sensitive to lenalidomide, and whether the compound could modulate cyclin D1/p27(KIP1) complexes in MCL cells. MCL primary samples and cell lines were analyzed for subcellular levels of cyclin D1/p27(KIP1) complexes by Western blot, immunohistochemistry, immunoprecipitation, and flow cytometry. Activity of lenalidomide in vitro and its effect on cyclin D1/p27(KIP1) complexes were evaluated by real-time PCR, immunoprecipitation, immunofluorescence, and Western blot. In vivo validation was carried out in a mouse xenograft model of human MCL. We found cyclin D1 and p27(KIP1) to be coordinately expressed in all the MCL samples tested. Immunoprecipitation analyses and siRNA assays suggested a direct role of cyclin D1 in the regulation of p27(KIP1) levels. The nuclear accumulation of both proteins correlated with MCL cell tumorigenicity in vivo, and sensitivity to lenalidomide activity in vitro and in vivo. Lenalidomide mechanism of action relied on cyclin D1 downregulation and disruption of cyclin D1/p27(KIP1) complexes, followed by cytosolic accumulation of p27(KIP1), cell proliferation arrest, apoptosis, and angiogenesis inhibition. These results highlight a mechanism of action of lenalidomide in MCL cases with increased tumorigenicity in vivo, which is mediated by the dissociation of cyclin D1/p27(KIP1) complexes, and subsequent proliferation blockade and apoptosis induction.

CDK2 and mTOR are direct molecular targets of isoangustone A in the suppression of human prostate cancer cell growth

Licorice extract which is used as a natural sweetener has been shown to possess inhibitory effects against prostate cancer, but the mechanisms responsible are poorly understood. Here, we report a compound, isoangustone A (IAA) in licorice that potently suppresses the growth of aggressive prostate cancer and sought to clarify its mechanism of action. We analyzed its inhibitory effects on the growth of PTEN-deleted human prostate cancer cells, in vitro and in vivo. Administration of IAA significantly attenuated the growth of prostate cancer cell cultures and xenograft tumors. These effects were found to be attributable to inhibition of the G1/S phase cell cycle transition and the accumulation of p27kip1. The elevated p27kip1 expression levels were concurrent with the decrease of its phosphorylation at threonine 187 through suppression of CDK2 kinase activity and the reduced phosphorylation of Akt at Serine 473 by diminishing the kinase activity of the mammalian target of rapamycin (mTOR). Further analysis using recombinant proteins and immunoprecipitated cell lysates determined that IAA exerts suppressive effects against CDK2 and mTOR kinase activity by direct binding with both proteins. These findings suggested that the licorice compound IAA is a potent molecular inhibitor of CDK2 and mTOR, with strong implications for the treatment of prostate cancer. Thus, licorice-derived extracts with high IAA content warrant further clinical investigation for nutritional sources for prostate cancer patients.

Geminin Is a Newly Identified Cks1 Interaction Partner and Overexpressed in a Murine Model During Lymphomagenesis

AbstractAbstract 1328Cks1 is an activator of the SCFSkp2 ubiquitin ligase complex that targets the cell cycle inhibitor p27Kip1 for degradation. Loss of Cks1 results in p27Kip1 accumulation and decreased proliferation. Cks1 expression is elevated in various B cell malignancies including Burkitt lymphoma and multiple myeloma. We have previously shown that loss of Cks1 results in elevated p27Kip1 levels and delayed tumor development in a mouse model of Myc-induced B cell lymphoma. Surprisingly, loss of Skp2 in the same mouse model also resulted in elevated p27Kip1 levels but exhibited no impact on tumor onset. Analysis of Cks1−/− murine embryonic fibroblasts (MEFs) unveiled a significant growth defect and comparison with double knock out Cks1−/−;p27Kip1−/− MEFs revealed a p27Kip1 independent defect at G1/S transition. These effects were associated with suppressed Cdk1 protein and Cdk2 activity in Cks1−/− cells.We hypothesized that Cks1 recruits further cell cycle regulators or facilitators of proliferation, which may explain the dramatic growth defect and the prolonged latency of lymphomagenesis upon loss of Cks1. Using Strep-Flag tagged Cks1 and tandem affinity purification followed by mass spectrometry we identified Geminin as a new Cks1 binding partner. Geminin is an inhibitor of rereplication and loss of Geminin leads to recurrent origin firing in S phase. The physical interaction of Cks1 and Geminin was present in cells of human and murine origin and was associated with Cdk2 binding to Geminin. Detailed analysis proved Cks1 dispensibel for cell cycle controlled Geminin degradation or phosphorylation.Surprisingly when mining public databases and RNA microarray data we found Geminin overexpressend in aggressive human lymphoma subtypes associated with high Ki-67 expression, and in murine Myc-induced lymphoma. Real time PCR revealed increased expression of Geminin during lymphomagenesis in Eμ-Myc mice, which correlated with protein expression. In conclusion, we established the physical Geminin-Cks1 protein interaction, present in human and murine cells, which could act in control of rereplication and genomic stability. Geminin is overexpressed in human and murine lymphoma revealing a potential role in lymphomagenesis. Disclosures:No relevant conflicts of interest to declare.

Epigenetic therapy with 3-deazaneplanocin A, an inhibitor of the histone methyltransferase EZH2, inhibits growth of non-small cell lung cancer cells

EZH2 (enhancer of zeste homolog 2) is the catalytic subunit of PRC2 (polycomb repressive complex 2), which mediates histone methyltransferase activity and functions as transcriptional repressor involved in gene silencing. EZH2 is involved in malignant transformation and biological aggressiveness of several human malignancies. We previously demonstrated that non-small cell lung cancers (NSCLCs) also overexpress EZH2 and that high expression of EZH2 correlates with poor prognosis. Growing evidence indicates that EZH2 may be an appropriate therapeutic target in malignancies, including NSCLCs. Recently, an S-adenosyl-l-homocysteine hydrolase inhibitor, 3-Deazaneplanocin A (DZNep), has been shown to deplete and inhibit EZH2. The aim of this study was to determine the effect of DZNep in NSCLC cells. Knockdown of EZH2 by small-interfering RNA (siRNA) resulted in decreased growth of four NSCLC cell lines. MTT assays demonstrated that DZNep treatment resulted in dose-dependent inhibition of proliferation in the NSCLC cell lines with a half maximal inhibitory concentration (IC50) ranging from 0.08 to 0.24μM. Immortalized but non-cancerous bronchial epithelial and fibroblast cell lines were less sensitive to DZNep than the NSCLC cell lines. Soft agarose assays demonstrated that anchorage-independent growth was also reduced in all three NSCLC cell lines that were evaluated using this assay. Flow cytometry analysis demonstrated that DZNep induced apoptosis and G1 cell cycle arrest in NSCLC cells, which was partially associated with cyclin A decrease and p27Kip1 accumulation. DZNep depleted cellular levels of EZH2 and inhibited the associated histone H3 lysine 27 trimethylation. These results indicated that an epigenetic therapy that pharmacologically targets EZH2 via DZNep may constitute a novel approach to treatment of NSCLCs.

Mevalonate Metabolism Regulates Basal Breast Cancer Stem Cells and Is a Potential Therapeutic Target

There is increasing evidence that breast tumors are organized in a hierarchy, with a subpopulation of tumorigenic cancer cells, the cancer stem cells (CSCs), which sustain tumor growth. The characterization of protein networks that govern CSC behavior is paramount to design new therapeutic strategies targeting this subpopulation of cells. We have sought to identify specific molecular pathways of CSCs isolated from 13 different breast cancer cell lines of luminal or basal/mesenchymal subtypes. We compared the gene expression profiling of cancer cells grown in adherent conditions to those of matched tumorsphere cultures. No specific pathway was identified to be commonly regulated in luminal tumorspheres, resulting from a minor CSC enrichment in tumorsphere passages from luminal cell lines. However, in basal/mesenchymal tumorspheres, the enzymes of the mevalonate metabolic pathway were overexpressed compared to those in cognate adherent cells. Inhibition of this pathway with hydroxy-3-methylglutaryl CoA reductase blockers resulted in a reduction of breast CSC independent of inhibition of cholesterol biosynthesis and of protein farnesylation. Further modulation of this metabolic pathway demonstrated that protein geranylgeranylation (GG) is critical to breast CSC maintenance. A small molecule inhibitor of the geranylgeranyl transferase I (GGTI) enzyme reduced the breast CSC subpopulation both in vitro and in primary breast cancer xenografts. We found that the GGTI effect on the CSC subpopulation is mediated by inactivation of Ras homolog family member A (RHOA) and increased accumulation of P27(kip1) in the nucleus. The identification of protein GG as a major contributor to CSC maintenance opens promising perspectives for CSC targeted therapy in basal breast cancer.

Effects of PD-1 signaling on the molecular control of T cell expansion (178.17)

Abstract Programmed death (PD)-1 is a potent T cell inhibitor critical for peripheral tolerance. Currently, it is unclear whether PD-1 has a global or a selective inhibitory effect on TCR signaling pathways involved in T cell expansion. It is also unclear how PD-1 affects the molecular machinery of cell cycle progression. Using primary human T cells we determined that PD-1 blocked cell cycle progression in the G1 phase. PD-1 did not alter the expression of G1 phase cyclins or cyclin-dependent kinases (Cdks) but, instead, suppressed transcription of the SCFSkp2 ubiquitin ligase that leads p27kip1 to degradation and resulted in accumulation of p27kip1. T cells receiving PD-1 signals displayed impaired Cdk2 activation and failed to phosphorylate Rb and Smad3 leading to suppression of E2F target genes but enhanced Smad3 transactivation. These events resulted in abrogation of the Cdk-activating phosphatase Cdc25A and upregulation of the Cdk4/6 inhibitor p15INK4. Suppression of Skp2 by PD-1 was mediated by inhibition of both PI3K/Akt and Ras/MEK/Erk pathways and was only partially reversed by IL-2, which restored activation of MEK/Erk but not Akt. In contrast to the inhibition of Ras and MEK/Erk pathway, PD-1 did not affect activation of Rap1, p38 and JNK MAP kinases. Thus, PD-1 selectively targets Ras/MEK/Erk and PI3K/Akt signaling to inhibit transcription of Skp2 and to activate Smad3 as an integral component of a pathway regulating blockade of cell cycle progression in T lymphocytes.

Abstract 3051: CDC28 protein kinase subunit 1B (CKS1B) is a potential therapeutic target in esophageal adenocarcinomas overexpressing the CDK inhibitor p21Cip1/Waf1

Abstract Background: The incidence of adenocarcinoma of the esophagus and gastroesophageal junction (EAC) has grown faster than any tumor type in the United States and westernized countries, outpacing the next closest cancer by almost 3 times. This rising incidence of EAC is associated with the increasing prevalence of obesity and gastroesophageal reflux disease (GERD). Due to the limited effect of current chemotherapy for this cancer, the search for new therapeutic targets is disparately needed. Chromosomal amplification is one mechanism by which cancer cells acquire and maintain neoplastic phenotype and genes that are amplified and overexpressed may represent potential therapeutic targets. Methods: DNA and RNA from a cohort of 116 patients were analyzed using SNP and mRNA expression microarrays respectively. Gene amplification was explored for correlation with expression and patient survival. Results: 25% of tumors harbor amplification at chromosome 1q which is significantly associated with poor survival. Although the amplicon is non-focal spanning almost the entire arm of chromosome 1q, only a subset of amplified genes (including The CDC28 protein kinase subunit 1B, CKS1B) are significantly overexpressed. CKS1B is an auxiliary protein required for ubiquitination and cytosomal degradation of the CDK inhibitors p27Kip1 and p21Cip1/Waf1 by the SCFSKP2 E3 ubiquitin ligase complex. Both p27Kip1 and p21Cip1/Waf1 inhibits CDK2/Cyclin E complex during the G1/S cell cycle transition. The endogenous protein expression of p27Kip1 is low among EAC cell lines. However the expression of p21Cip1/Waf1 is differential being very high in a subset of cells and low or undetectable in others. Knocking down CKS1B by siRNA in six EAC cell lines results in accumulation of p27 Kip1 with little or no effect on p21Cip1/Waf1 expression. CKS1B knockdown inhibits monolayer cell proliferation only in cells with high endogenous expression of p21Cip1/Waf1. Conclusion: We suggest that the combined action of accumulation of p27 Kip1 and the endogenously highly expressed p21Cip1/Waf1 triggers the response to CKS1B knockdown in EAC cell lines. We thus propose the potential use of CKS1B as a novel therapeutic target in esophageal adenocarcinomas with high p21Cip1/Waf1 expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3051. doi:1538-7445.AM2012-3051