G1-S Transition Research Articles

Effects of gene silencing of CypB on gastric cancer cells.

To determine the effect of gene silencing of cyclophilin B (CypB) on growth and proliferation of gastric cancer cells. CypB siRNA lentivirus (LV-CypB-si) and control lentivirus (LV-si-con) were produced. CypB expression in gastric cancer cell lines was detected by Western blot. BGC823 and SGC7901 cells were chosen to be infected with LV-si-con and LV-CypB-si, and stable transfectants were isolated. The cell groups transfected with LV-CypB-siRNA, LV-siRNA-con and transfected no carrier were served as the experimental group, the implicit control group and the blank control group respectively. MTT and colony formation assays were used to examine the effect of CypB on the cell growth and proliferation in vitro. Cell cycle was analyzed with flow cytometry. The expression of VEGFR of BGC823-si and SGC7901-si was detected by Western blot. Gene silencing of CypB can inhibit gastric cancer cell growth, proliferation, cell cycle progress and tumorigenesis. CypB expression level was obviously higher in SGC7901 and BGC823 than MKN28 and GES. These two cell lines were infected with LV-si-con and LV-CypB-si respectively. MTT and cloney formation assays showed a significantly decreased rate of cell proliferation from the forth day or the fifth day in cells transfected with LV-CypB-si (P<0.05). Down-regulation of CypB resulted in slightly decreased percentage of S phase and increased percentage of G1 (P<0.05). These findings indicated that CypB could promote the G1-S transition of gastric cancer cell. In addition, the expression of VEGF of BGC823 and SGC7901 transfected with CypB siRNA was reduced in comparison with the implicit control group and the blank control group. Gene silencing of CypB decreases gastric cancer cells proliferation and in vivo tumorigenesis. These findings indiccate CypB could be a potential biomarker and therapeutic target for gastric cancer.

Optimization of Enzyme Dilution Buffer and Reaction Buffer for Vaccinia H-1 Related Wild Enzyme Assay and Its Stability at 4°C

A human dual specific phosphates (DUSP), Vaccinia H-1 related wild enzyme (VHR), dephosphorylates both phosphotyrosine and phosphoserine/phosphothreonine residue of a protein. VHR is considered as a promising therapeutic target for cancer because the cells lacking VHR are arrested at the G1-S and G2-M transitions of the cell cycle with a decreased telomerase activity. VHR being a therapeutic target for cancer is crucial to know about its stability and its assay procedure conditions. This study was conducted to verify the viability of VHR enzyme at 4°C. Protein concentration and specific activity were calculated from Bradford method and p-nitrophenylphosphate (pNPP) assay by measuring the absorbance at 595 nm and 405 nm respectively in each respective day. The absorbance showed invariable difference in protein concentration and specific activity from starting to final days. Buffers like enzyme dilution and reaction buffers played significant role in VHR enzyme stability and activity. To find out the correct buffer components for carrying out the VHR enzyme assay, several experiments were carried out by using variable constituents in enzyme dilution buffer and reaction buffer. The present study revealed that 3- component buffer system without thiol i.e. Dithiothreitol (DTT) or β-Mercaptoethanol (β-ME) as a reaction buffer and 2-N-morpholino ethanesulfonic acid (MES) buffer with DTT as an enzyme dilution buffer demonstrated invariably different in the stability throughout the experiment.DOI: http://dx.doi.org/njst.v15i2.12127Nepal Journal of Science and Technology Vol. 15, No.2 (2014) 117-122

Vascular endothelial growth factor receptor 3 controls neural stem cell activation in mice and humans.

SUMMARYNeural stem cells (NSCs) continuously produce new neurons within the adult mammalian hippocampus. NSCs are typically quiescent but activated to self-renew or differentiate into neural progenitor cells. The molecular mechanisms of NSC activation remain poorly understood. Here, we show that adult hippocampal NSCs express vascular endothelial growth factor receptor (VEGFR) 3 and its ligand VEGF-C, which activates quiescent NSCs to enter the cell cycle and generate progenitor cells. Hippocampal NSC activation and neurogenesis are impaired by conditional deletion of Vegfr3 in NSCs. Functionally, this is associated with compromised NSC activation in response to VEGF-C and physical activity. In NSCs derived from human embryonic stem cells (hESCs), VEGF-C/VEGFR3 mediates intracellular activation of AKT and ERK pathways that control cell fate and proliferation. These findings identify VEGF-C/VEGFR3 signaling as a specific regulator of NSC activation and neurogenesis in mammals.

MicroRNA-3127 promotes cell proliferation and tumorigenicity in hepatocellular carcinoma by disrupting of PI3K/AKT negative regulation.

Recent studies have shown that multiple phosphatases deactivate the PI3K/AKT signaling pathway. Here we demonstrated that, by suppressing multiple phosphatases, miR-3127 promotes growth of hepatocellular carcinoma (HCC). Our study also reveals clinical significance of miR-3127 expression in HCC patients. MiR-3127 expression was markedly upregulated in HCC tissues and cells. Furthermore, high miR-3127 expression was associated with an aggressive phenotype and poor prognosis. MiR-3127 overexpression promoted HCC cell proliferation in vitro and tumor growth in vivo. Also, miR-3127 accelerated G1-S transition by activating AKT/ FOXO1 signaling, by directly targeting the 3' untranslated regions (3`UTR) of pleckstrin homology domain leucine-rich repeat protein phosphatase 1/2 (PHLPP1/2), inositol polyphosphate phosphatase 4A (INPP4A), and inositol polyphosphate-5-phosphatase J (INPP5J) mRNA, repressing their expression. In agreement, the miRNA antagonist antagomir-3127 suppressed HCC cell proliferation and tumor growth by inhibiting the AKT/FOXO1 signaling. Taken together, these findings suggest that silencing miR-3127 might be a potential therapeutic strategy.

Blockage of PTPRJ promotes cell growth and resistance to 5-FU through activation of JAK1/STAT3 in the cervical carcinoma cell line C33A.

Gene therapy is a promising therapeutic approach for chemoresistant cervical cancers. Therapeutic interventions targeting the key factors contributing to the initiation and progression of cervical cancer may be a more effective treatment strategy. In the present study, we firstly determined the expression of protein tyrosine phosphatase receptor J (PTPRJ) in 8-paired human cervical tumor and non-tumor tissues. We observed a striking downregulation of PTPRJ in the human cervical tumor tissues. Next, we investigated the roles and the function mechanism of PTPRJ in the human cervical carcinoma cell line C33A by loss- and gain-of-function experiments. Our study indicated that C33A cells with loss of PTPRJ expression showed a significantly increased cell viability, rising growth and migration rate, as well as a G1-S transition. We obtained the opposite results when we overexpressed PTPRJ in C33A cells. Our further study indicated that PTPRJ levels were highly correlated with cell survival when the C33A cells were treated with 5-fluorouracil (5-FU), an important chemotherapeutic agent for cervical cancer. In addition, the signaling pathway screening assay showed an obvious alteration of the Janus kinase 1/signal transducer and activator of transcription 3 (JAK1/STAT3) pathway. PTPRJ negatively regulated the activation of the JAK1/STAT3 pathway by decreasing the phosphorylation levels of JAK1 and STAT3. In addition, PTPRJ also regulated the expression of the downstream factors of STAT3, such as cyclin D, Bax, VEGF and MMP2. Our results suggest that PTPRJ may be a promising gene therapy target and its therapeutic potential can be fulfilled when used alone, or in combination with other anticancer agents.

A role of Candida albicans CDC4 in the negative regulation of biofilm formation.

The CDC4 gene is nonessential in Candida albicans and plays a role in suppressing filamentous growth, in contrast to its homologues, which are involved in the G1-S transition of the cell cycle. While characterizing the function of C. albicans CDC4 (CaCDC4), we found that the loss of CaCDC4 resulted in a reduction in cell flocculation, indicating a possible role for CaCDC4 in biofilm formation. To elucidate the role of CaCDC4 in biofilm formation, Cacdc4 null mutant strains were constructed by using the mini-Ura-blaster method. To create a CaCDC4 rescued strain, the plasmid p6HF-ACT1p-CaCDC4 capable of constitutively expressing CaCDC4 was introduced into the Cacdc4 homozygous null mutant. To determine the biofilm formation ability, an in vitro XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium-5-carboxanilide) reduction assay was used. Compared with the parental auxotrophic strain BWP17, the Cacdc4 homozygous null mutant was able to enhance biofilm formation significantly. This enhancement of biofilm formation in the Cacdc4 homozygous null mutant could be reversed by constitutively expressing CaCDC4. We conclude that CaCDC4 has a role in suppressing biofilm formation in C. albicans.

The Cell Cycle Regulators CDK4, CDK6 and Cyclin D3 As Well As the Histone Methyltransferases MLL2 and MLL4 Constitute NF-E2 Target Genes That Participate in a Novel Regulatory Loop

Background: Expression of the transcription factor and epigenetic modulator “nuclear factor erythroid-2” (NF-E2) is aberrantly elevated in patients with Myeloproliferative Neoplasms (MPN). We have shown that NF-E2 overexpression in a murine model causes a MPN phenotype. In addition, we have identified in-del mutations in the NF-E2 gene in MPN patients, which, despite having lost the ability to bind DNA, nonetheless increase the activity of wild type NF-E2. These mutants, which are acquired secondary to the JAK2V617F mutation, confer a proliferative advantage beyond that provided by the JAK2V617F alone. In all patients analyzed, cells carrying both mutations displayed clonal dominance, vastly outnumbering those carrying JAK2V617Falone. Mutant NF-E2 causes an increase in the number of cells entering S-phase mediated by increased expression of the cell cycle regulators CDK4, CDK6 and CyclinD3 that promote the G1-S transition. However, it is not known whether these cell cycle regulators constitute direct NF-E2 target genes or whether the proliferative effect is indirect. Moreover, additional downstream pathways by which this augmented transcription factor activity exerts its effects remain unknown.Aims: We therefore investigated whether CDK4, CDK6 and Cyclin D3 constitute direct NF-E2 target genes. Moreover, we identified additional novel NF-E2 targets in an effort to delineate the molecular mechanism by which increased NF-E2 activity contributes to MPN pathophysiology.Methods: We used ChIP and luciferase reporter assays as well as qRT-PCR and Western Blot to identify and characterize novel NF-E2 targets.Results: Our data show that both the cell cycle regulators CDK4, CDK6 and CyclinD3 as well as the histone methyltransferases MLL2 and MLL4 constitute direct NF-E2 target genes, as the transcription factor binds these genes in vivo.ChIP analysis of HEL cells shows NF-E2 binding to a site in the CDK4 promoter, four sites in the CDK6 and the Cyclin D3 locus each, to the MLL4 promoter as well as to three sites in the MLL2 locus, establishing these five genes as direct NF-E2 targets. Activity of NF-E2 on the MLL4 promoter was confirmed by reporter gene assays. Expression of both the cell cycle regulators and the methyltransferases was statistically significantly elevated in bone marrow from NF-E2 overexpressing mice. Most importantly, quantitative RT-PCR showed significantly increased mRNA expression of all five target genes in primary cells of patients with polycythemia vera (PV) compared to healthy controls. Hence, the expression of the cell cycle regulators and the epigenetically active enzymes is augmented in PV patients. Corresponding to the increased methyltransferase activity, primary cells from PV patients showed a significant elevation in global H3K4m1 levels, a chromatin mark conferred by the MLL proteins.The MLL2 protein has been previously shown to interact with NF-E2. NF-E2 serves as a scaffold and is required both for recruitment of MLL2 to the beta-globin locus as well as for chromatin remodeling at these sites. Here we show that MLL2 binds the CDK6 locus at the same sites as NF-E2, strongly suggesting that NF-E2 likewise acts to direct MLL2 to the CDK6 gene.Our data establish a novel interaction network where NF-E2 both regulates the expression levels of MLL2 while at the same time modulating its epigenetic activity at NF-E2 target genes, which include critical cell cycle regulators.Summary / Conclusions: Here we show that the cell cycle regulators CDK4, CDK6 and Cyclin D3 as well as the histone methyltransferases MLL2 and MLL4 constitute novel NF-E2 target genes. We propose that elevated NF-E2 levels in MPN patients contribute to disease pathophysiology by participating in a novel regulatory loop, which both enhances transcription and alteres histone methylation leading to increased proliferation. These data provide a molecular basis for pre-clinical investigation into the effects of CDK 4/6 inhibitors as well as of histone methyltransferase inhibitors on MPN cell biology. DisclosuresNo relevant conflicts of interest to declare.

Abstract A43: RUNX3 and pRB form a complex and regulate restriction-point commitment

Abstract Throughout the cell cycle, a cell monitors cumulative exposure to specific signals over time and makes a critical decision to pass through the restriction (R) point. Although the R-point decision is fundamental to normal differentiation and G1-S transition, the mechanism for the decision has been poorly understood. RUNX3 functions as a tumor suppressor and is frequently inactivated by DNA hyper-methylation in the stomach, bladder, colon, and lung. Recently, we have shown that adenovirus-Cre infected LSL-K-RasG12D mice induced lung adenocarcinoma (ADC) in prolonged latency (median survival, 220 days) and Runx3f/f/LSL-K-RasG12D mice rapidly induced ADC (median survival, 79 days), indicating a strong tumor suppressor activity of Runx3 against oncogenic K-Ras (Lee et., 2013. Cancer Cell. 2013 Vol. 24, pp 603-616). In the present study, we found that targeted disruption of Runx3 results in a delay in pRB phosphorylation and cell cycle progression through the R-point in mouse embryonic fibroblast cells. During the R-point interval, RUNX3 transiently formed a complex with pRB and BRD2 and induced Cdkn1a (p21Waf1/Cip1/Sdi1; p21). The complex was dissociated when by Cyclin D1 which was induced 4 hours after serum stimulation and p21 expression was turned off. However, oncogenic K-Ras maintained the complex. These results identify the series of molecular events associated with R-point commitment and provide an insight into the relationship between R-point commitment and oncogene surveillance. Note: This abstract was not presented at the conference. Citation Format: Xin-Zi Chi, You-Soub Lee, Jung-Won Lee, You-Mie Lee, Yoshiaki Ito, Suk-Chul Bae. RUNX3 and pRB form a complex and regulate restriction-point commitment. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A43. doi: 10.1158/1557-3125.RASONC14-A43

Human RAD6 promotes G1-S transition and cell proliferation through upregulation of cyclin D1 expression.

Protein ubiquitinylation regulates protein stability and activity. RAD6, an E2 ubiquitin-conjugating enzyme, which that has been substantially biochemically characterized, functions in a number of biologically relevant pathways, including cell cycle progression. In this study, we show that RAD6 promotes the G1-S transition and cell proliferation by regulating the expression of cyclin D1 (CCND1) in human cells. Furthermore, our data indicate that RAD6 influences the transcription of CCND1 by increasing monoubiquitinylation of histone H2B and trimethylation of H3K4 in the CCND1 promoter region. Our study presents, for the first time, an evidence for the function of RAD6 in cell cycle progression and cell proliferation in human cells, raising the possibility that RAD6 could be a new target for molecular diagnosis and prognosis in cancer therapeutics.

KSHV viral cyclin interferes with T-cell development and induces lymphoma through Cdk6 and Notch activation in vivo

Kaposi's sarcoma herpesvirus (KSHV)-encoded v-cyclin, a homolog of cellular cyclin D2, activates cellular CDK6, promotes G1-S transition of the cell cycle, induces DNA damage, apoptosis, autophagy and is reported to have oncogenic potential. Here we show that in vivo expression of v-cyclin in the B- and T-cell lymphocyte compartments results in a markedly low survival due to high penetrance of early-onset T-cell lymphoma and pancarditis. The v-cyclin transgenic mice have smaller pre-tumorigenic lymphoid organs, showing decreased cellularity, and increased proliferation and apoptosis. Furthermore, v-cyclin expression resulted in decreased amounts of CD3-expressing mature T-cells in the secondary lymphoid organs concurrent with alterations in the T-cell subpopulations of the thymus. This suggests that v-cyclin interferes with normal T-cell development. As the Notch pathway is recognized for its role in both T-cell development and lymphoma initiation, we addressed the role of Notch in the v-cyclin-induced alterations. Fittingly, we demonstrate induction of Notch3 and Hes1 in the pre-tumorigenic thymi and lymphomas of v-cyclin expressing mice, and show that lymphoma growth and viability are dependent on activated Notch signaling. Notch3 transcription and growth of the lymphomas was dependent on CDK6, as determined by silencing of CDK6 expression or chemical inhibition, respectively. Our work here reveals a viral cyclin-CDK6 complex as an upstream regulator of Notch receptor, suggesting that cyclins can play a role in the initiation of Notch-dependent lymphomagenesis.

Metformin repositioning as antitumoral agent: selective antiproliferative effects in human glioblastoma stem cells, via inhibition of CLIC1-mediated ion current.

Epidemiological and preclinical studies propose that metformin, a first-line drug for type-2 diabetes, exerts direct antitumor activity. Although several clinical trials are ongoing, the molecular mechanisms of this effect are unknown. Here we show that chloride intracellular channel-1 (CLIC1) is a direct target of metformin in human glioblastoma cells. Metformin exposure induces antiproliferative effects in cancer stem cell-enriched cultures, isolated from three individual WHO grade IV human glioblastomas. These effects phenocopy metformin-mediated inhibition of a chloride current specifically dependent on CLIC1 functional activity. CLIC1 ion channel is preferentially active during the G1-S transition via transient membrane insertion. Metformin inhibition of CLIC1 activity induces G1 arrest of glioblastoma stem cells. This effect was time-dependent, and prolonged treatments caused antiproliferative effects also for low, clinically significant, metformin concentrations. Furthermore, substitution of Arg29 in the putative CLIC1 pore region impairs metformin modulation of channel activity. The lack of drugs affecting cancer stem cell viability is the main cause of therapy failure and tumor relapse. We identified CLIC1 not only as a modulator of cell cycle progression in human glioblastoma stem cells but also as the main target of metformin's antiproliferative activity, paving the way for novel and needed pharmacological approaches to glioblastoma treatment.

WNT5A modulates cell cycle progression and contributes to the chemoresistance in pancreatic cancer cells

Although there are many studies on the mechanism of chemoresistance in cancers, studies on the relations between WNT5A and chemoresistance in pancreatic cancer are rare. The present study was to examine the role of WNT5A in the regulation of cell cycle progression and in chemo-resistance in pancreatic cancer tissues and cell lines. Fresh pancreatic cancer and paracarcinoma tissues were obtained from 32 patients. The expressions of WNT5A, AKT/p-AKT and Cyclin D1 were detected by immunohistochemistry, and the correlation between WNT5A expression and clinicopathological characteristics was analyzed. The relationship between WNT5A expression and gemcitabine resistance was studied in PANC-1 and MIAPaCa2 cell lines. The effect of WNT5A on the regulation of cell cycle and gemcitabine cytotoxicity were investigated. The associations among the expressions of p-AKT, Cyclin D1 and WNT5A were also analyzed in cell lines and the effect of WNT5A on restriction-point (R-point) progression was evaluated. WNT5A, p-AKT and Cyclin D1 were highly expressed in pancreatic cancer tissues, and the WNT5A expression was correlated with the TNM stages. In vitro, WNT5A expression was associated with gemcitabine chemoresistance. The percentage of cells was increased in G0/G1 phase and decreased in S phase after knockdown of WNT5A in PANC-1. WNT5A promoted Cyclin D1 expression through phosphorylation of AKT which consequently enhanced G1-S transition and gemcitabine resistance. Furthermore, WNT5A enhanced the cell cycle progression toward R-point through regulation of retinoblastoma protein (pRb) and pRb-E2F complex formation. WNT5A induced chemoresistance by regulation of G1-S transition in pancreatic cancer cells. WNT5A might serve as a predictor of gemcitabine response and as a potential target for tumor chemotherapy.

Abstract 2753: Ormeloxifene inhibits cervical cancer cell growth through intrinsic apoptotic pathway

Abstract Background: Cervical cancer (CxCa) remains the fourth leading cause of cancer related death among women worldwide. Cervical cancer is mainly (∼ 99.7%) derived from high risk Human papillomavirus (HR HPV). HPV E6/E7 oncoproteins interfere with p53 and pRb (retinoblastoma) cell cycle regulatory proteins and hinder their efficacy of controlling cell growth. Advanced stage cervical cancer is difficult to treat and patients diagnosed with metastatic disease have a poor survival rate of only 16%. Therefore, there is an urgent need to develop newer treatment modalities. Ormeloxifene is a non-hormonal, anti-estrogen, oral contraceptive for human use that effectively suppresses the proliferation of decidual cells in the endometrium to inhibit egg implantation. Growing evidences also suggest that ormeloxifene has anti-cancerous properties in breast, head and neck, and chronic myeloid leukemia cancers. Hence, considering its strong anti-proliferative/cancerous effect, we hypothesized that ormeloxifene may also inhibit the growth of cervical cancer cells and may suppress the expression of E6 and E7 HPV oncoproteins. Methods: We performed in vitro anti-cancer studies with ormeloxifene (dose range: 5 µM to 25 µM) in the HPV positive cervical cancer cell lines (Caski and SiHa) by utilizing MTS and colony formation assays. To further determine the effects of ormeloxifene on the cellular and molecular level, immunoblotting, flow cytometry and real time PCR analyses were performed using the Caski cell line model. Results: Ormeloxifene effectively inhibited cervical cancer cell growth as determined by proliferation and colony formation assays in a dose dependent manner. Ormeloxifene decreased mitochondrial membrane potential and induced apoptosis as shown by flow cytometry analysis of cells stained with TMRE (tetramethylrhodamine, ethyl ester) and 7AAD/Annexin V, respectively. Apoptosis was also confirmed by immunoblotting for cleaved PARP (Poly ADP ribose polymerase), Caspase 3 and Caspase 9. Additionally, ormeloxifene treatment arrested cell cycle in G1-S transition and decreased the expression of p-Akt while total Akt remained the same. Specifically relevant to cervical cancer, ormeloxifene decreased the expression of HPV E6/E7 at mRNA and protein levels as determined by quantitative PCR analysis and immunoblotting, respectively. Interestingly, ormeloxifene restored the levels of p53, Rb and PTPN 13 (Protein tyrosine Phosphatase non receptor type 13) tumor suppressor proteins as shown by immunoblotting. Conclusion: Our findings suggest that ormeloxifene inhibits cervical cancer cell growth and decreases mitochondrial membrane potential which clearly demonstrates that apoptosis is induced via intrinsic pathway. Importantly, ormeloxifene downregulates the expression of HPV E6/E7 oncoproteins. Thus, ormeloxifene appears to be a novel treatment modality for cervical cancer. Citation Format: Neeraj Chauhan, Mohd S. Zaman, Murali M. Yallapu, Diane M. Maher, Mara C. Ebeling, Subhash C. Chauhan, Meena Jaggi. Ormeloxifene inhibits cervical cancer cell growth through intrinsic apoptotic pathway. [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 2753. doi:10.1158/1538-7445.AM2014-2753

RB family tumor suppressor activity may not relate to active silencing of E2F target genes.

The retinoblastoma protein pRB and its two homologs p130 and p107 form the family of pocket proteins and play a major role in cell-cycle regulation and suppression of human and mouse tumorigenesis. Pocket proteins regulate the activity of E2F transcription factors during G1-S transition. Two mechanisms have been described: (i) pocket protein binding blocks the transactivation domain of activator E2Fs, inhibiting E2F-dependent transcription and (ii) E2F-bound pocket proteins can recruit chromatin remodeling proteins containing an LxCxE motif (x encoding any amino acid), resulting in active repression of E2F target genes. To investigate the importance of pRB's LxCxE-interacting motif in cell-cycle control and tumor suppression, we generated mouse embryonic fibroblasts and mice expressing a mutant pRB protein carrying an asparagine for phenylalanine substitution at position 750, abrogating LxCxE binding. Because p130 may compensate for loss of pRB, we studied pRB(N750F) activity in the presence and absence of p130. The pRB-LxCxE interaction was not required for cell-cycle arrest upon mitogen deprivation and cell-cell contact, but did contribute to RAS(V12)- and radiation-induced cell-cycle arrest. Remarkably, the pRB-LxCxE interaction was not required for suppression of in vitro and in vivo transformation, even in the absence of p130. These results indicate that pRB's tumor suppressor activity is not effectuated by active silencing of E2F target genes, but rather by regulation of activator E2Fs or another unidentified mechanism. Furthermore, the in vitro response of pocket protein-perturbed cells to mitogen deprivation and cell-cell contact seems a better predictor of tumor development than the response to ectopic RAS(V12) expression. Cancer Res; 74(18); 5266-76. ©2014 AACR.

Targeting protein synthesis in cancer cells.

The G1 and S phases of the mitotic cell cycle normally insure that the parental cell attains a sufficient mass so as each daughter cell will have a size identical to that of the parental cell. This implies that before division cells must double their protein content, a process achieved through increase in protein synthesis. The most regulated step of protein synthesis is the initiation of mRNA translation into protein. Ribosome recruitment at the mRNA 5’ end is actually controlled by the mRNA 5’ cap binding protein eIF4E (eukaryotic translation initiation factor 4E) whose activity is inhibited by the hypophosphorylated forms of 4E-BPs (eIF4E-binding proteins 1 and 2) [1]. Upon mitogenic stimuli, mTOR phosphorylates 4E-BP1 and 4E-BP2 and the protein kinases S6K1 and S6K2 (S6Ks). 4E-BPs phosphorylation releases eIF4E which can interact with eIF4G itself bound to the RNA helicase eIF4A and to eIF3. Activated S6Ks phosphorylate eIF3 [2] and translation initiation is enhanced via eIF3-dependent recruitment of the 40S small ribosomal subunit and its subsequent joining with the large 60S subunit at the AUG initiator codon (Fig. 1). Consistent with a role of mTOR in G1-S transition,

Molecular pathways: CDK4 inhibitors for cancer therapy.

Unrestrained growth is the hallmark of cancer, and disrupted cell-cycle regulation is, therefore, common. CDK4 is the key regulator of the G1-S transition. In complex with cyclin D, CDK4 phosphorylates retinoblastoma protein (Rb) and drives cell-cycle progression, a process inhibited by p16. The p16-CDK4-cyclin D-Rb is aberrant in the majority of cancers and is, thus, a logical target for anticancer therapy. Previous attempts to block CDK4 with nonselective cyclin-dependent kinase (CDK) inhibitors led to toxicity and little efficacy. However, the recent development of selective CDK4 inhibitors launched the first successful efforts to target the pathway for cancer therapy. Three oral selective CDK4 inhibitors have entered clinical trials: palbociclib (PD0332991), LEE011, and LY2835219. CDK4 inhibitors have in vitro activity against a broad range of cancers and in patients have shown antitumor activity in breast cancer, lymphoma, sarcoma, and other tumors. Major efforts are under way to develop biomarkers of response, understand potential mechanisms of resistance, and develop rational combinations of CDK4 inhibitors with chemotherapy and other targeted drugs.

Zn-driven discovery of a hydrothermal vent fungal metabolite clavatustide C, and an experimental study of the anti-cancer mechanism of clavatustide B.

A naturally new cyclopeptide, clavatustide C, was produced as a stress metabolite in response to abiotic stress elicitation by one of the hydrothermal vent fluid components Zn in the cultured mycelia of Aspergillus clavatus C2WU, which were isolated from Xenograpsus testudinatus. X. testudinatus lives at extreme, toxic habitat around the sulphur-rich hydrothermal vents in Taiwan Kueishantao. The known compound clavatustide B was also isolated and purified. This is the first example of a new hydrothermal vent microbial secondary metabolite produced in response to abiotic Zn treatment. The structures were established by spectroscopic means. The regulation of G1-S transition in hepatocellular carcinoma cell lines by clavatustide B was observed in our previous study. The purpose of the present study was to verify these results in other types of cancer cell lines and elucidate the possible molecular mechanism for the anti-cancer activities of clavatustide B. In different human cancer cell lines, including pancreatic cancer (Panc-1), gastric cancer (MGC-803), colorectal cancer (SW-480), retinoblastoma (WERI-Rb-1) and prostate cancer (PC3), clavatustide B efficiently suppressed cell proliferations in a dose-dependent manner. Although different cancer cell lines presented variety in Max effect dose and IC50 dose, all cancer cell lines showed a lower Max effect dose and IC50 dose compared with human fibroblasts (hFB) (p < 0.05). Moreover, significant accumulations in G1 phases and a reduction in S phases (p < 0.05) were observed under clavatustide B treatment. The expression levels of 2622 genes including 39 cell cycle-associated genes in HepG2 cells were significantly altered by the treatment with 15 μg/mL clavatustide B after 48 h. CCNE2 (cyclin E2) was proved to be the key regulator of clavatustide B-induced G1-S transition blocking in several cancer cell lines by using real-time PCR.

Adjuvant palbociclib (P) plus endocrine therapy (ET) for hormone receptor positive (HR+) breast cancer: A phase II feasibility study.

TPS654 Background: Cell cycle inhibition is a target of interest for novel cancer therapeutics. P is an orally active inhibitor of CDK4/6, causing cell cycle arrest at the G1-S transition. In an in...

ERRβ signalling through FST and BCAS2 inhibits cellular proliferation in breast cancer cells

Background:The overexpression of oestrogen-related receptor-β (ERRβ) in breast cancer patients is correlated with improved prognosis and longer relapse-free survival, and the level of ERRβ mRNA is inversely correlated with the S-phase fraction of cells from breast cancer patients.Methods:Chromatin immunoprecipitation (ChIP) cloning of ERRβ transcriptional targets and gel supershift assays identified breast cancer amplified sequence 2 (BCAS2) and Follistatin (FST) as two important downstream genes that help to regulate tumourigenesis. Confocal microscopy, co-immunoprecipitation (CoIP), western blotting and quantitative real-time PCR confirmed the involvement of ERRβ in oestrogen signalling.Results:Overexpressed ERRβ induced FST-mediated apoptosis in breast cancer cells, and E-cadherin expression was also enhanced through upregulation of FST. However, this anti-proliferative signalling function was challenged by ERRβ-mediated BCAS2 upregulation, which inhibited FST transcription through the downregulation of β-catenin/TCF4 recruitment to the FST promoter. Interestingly, ERRβ-mediated upregulation of BCAS2 downregulated the major G1-S transition marker cyclin D1, despite the predictable oncogenic properties of BCAS2.Interpretation:Our study provides the first evidence that ERRβ, which is a coregulator of ERα also acts as a potential tumour-suppressor molecule in breast cancer. Our current report also provides novel insights into the entire cascade of ERRβ signalling events, which may lead to BCAS2-mediated blockage of the G1/S transition and inhibition of the epithelial to mesenchymal transition through FST-mediated regulation of E-cadherin. Importantly, matrix metalloprotease 7, which is a classical mediator of metastasis and E-cadherin cleavage, was also restricted as a result of ERRβ-mediated FST overexpression.

Arabidopsis Retinoblastoma-related and Polycomb group proteins: cooperation during plant cell differentiation and development

RETINOBLASTOMA (RB) is a tumour suppressor gene originally discovered in patients that develop eye tumours. The pRb protein is now well established as a key cell-cycle regulator which suppresses G1-S transition via interaction with E2F-DP complexes. pRb function is also required for a wide range of biological processes, including the regulation of stem-cell maintenance, cell differentiation, permanent cell-cycle exit, DNA repair, and genome stability. Such multifunctionality of pRb is thought to be facilitated through interactions with various binding partners in a context-dependent manner. Although the molecular network in which RB controls various biological processes is not fully understood, it has been found that pRb interacts with transcription factors and chromatin modifiers to either suppress or promote the expression of key genes during the switch from cell proliferation to differentiation. RETINOBLASTOMA-RELATED (RBR) is the plant orthologue of RB and is also known to negatively control the G1-S transition. Similar to its animal counterpart, plant RBR has various roles throughout plant development; however, much of its molecular functions outside of the G1-S transition are still unknown. One of the better-characterized molecular mechanisms is the cooperation of RBR with the Polycomb repressive complex 2 (PRC2) during plant-specific developmental events. This review summarizes the current understanding of this cooperation and focuses on the processes in Arabidopsis in which the RBR-PRC2 cooperation facilitates cell differentiation and developmental transitions.