Growth Repression Research Articles

MiR-3188 regulates nasopharyngeal carcinoma proliferation and chemosensitivity through a FOXO1-modulated positive feedback loop with mTOR-p-PI3K/AKT-c-JUN.

The biological role of miR-3188 has not yet been reported in the context of cancer. In this study, we observe that miR-3188 not only reduces cell-cycle transition and proliferation, but also significantly prolongs the survival time of tumour-bearing mice as well as sensitizes cells to 5-FU. Mechanistic analyses indicate that miR-3188 directly targets mTOR to inactivate p-PI3K/p-AKT/c-JUN and induces its own expression. This feedback loop further suppresses cell-cycle signalling through the p-PI3K/p-AKT/p-mTOR pathway. Interestingly, we also observe that miR-3188 direct targeting of mTOR is mediated by FOXO1 suppression of p-PI3K/p-AKT/c-JUN signalling. In clinical samples, reduced miR-3188 is an unfavourable factor and negatively correlates with mTOR and c-JUN levels but positively correlates with FOXO1 expression. Our studies demonstrate that as a tumour suppressor, miR-3188 directly targets mTOR to stimulate its own expression and participates in FOXO1-mediated repression of cell growth, tumorigenesis and NPC chemotherapy resistance.

Combination with γ-secretase inhibitor prolongs treatment efficacy of BRAF inhibitor in BRAF-mutated melanoma cells

Oncogenic triggering of the MAPK pathway in melanocytes results in senescence, and senescence escape is considered as one critical step for melanocytic transformation. In melanoma, induction of a senescent-like state by BRAF-inhibitors (BRAFi) in a fraction of treated cells – instead of killing – contributes to the repression of tumor growth, but may also provide a source for relapse. Here, we demonstrate that NOTCH activation in melanocytes is not only growth-promoting but it also protects these cells against oncogene-induced senescence. In turn, treatment of melanoma cells with an inhibitor of the NOTCH-activating enzyme γ-secretase led to induction of a senescent-like status in a fraction of the cells but overall achieved only a moderate inhibition of melanoma cell growth. However, combination of γ-secretase inhibitor (GSI) with BRAFi markedly increased the treatment efficacy particularly in long-term culture. Moreover, even melanoma cells starting to regrow after continuous BRAFi treatment – the major problem of BRAFi therapy in patients – can still be affected by the combination treatment. Thus, combining GSI with BRAFi increases the therapeutic efficacy by, at least partially, prolonging the senescent-like state of treated cells.

MiR-195 inhibits tumor growth and angiogenesis through modulating IRS1 in breast cancer.

Angiogenesis has been found as an attractive target for drug therapy as it is necessary for tumor growth. Accumulating evidences show that microRNAs (miRNAs), which are a group of highly conserved, single-stranded, short non-coding RNAs, play important roles through directly targeting angiogenic factors and protein kinases. The purpose of this study is to investigate the role of miR-195 in breast cancer development and angiogenesis through targeting IRS1. We show that miR-195 is inversely related with Insulin receptor substrate 1 (IRS1) in both breast cancer cells and breast cancer tissues. Induction of miR-195 could suppress IRS1 protein expression through binding to its 3'UTR regions either by transfection with miR-195 oligo or by infection with lentivirus encoding miR-195 gene. Moreover, re-expression of IRS1 reverses miR-195-mediated repression of tumor cell growth and miR-195 inhibits tumor angiogenesis through suppressing IRS1-VEGF axis. These data suggest that miR-195 mimics are potential therapeutic agents for breast cancer diagnose.

Critical Roles of Vacuolar Invertase in Floral Organ Development and Male and Female Fertilities Are Revealed through Characterization of GhVIN1-RNAi Cotton Plants.

Seed number and quality are key traits determining plant fitness and crop yield and rely on combined competence in male and female fertilities. Sucrose metabolism is central to reproductive success. It remains elusive, though, how individual sucrose metabolic enzymes may regulate the complex reproductive processes. Here, by silencing vacuolar invertase (VIN) genes in cotton (Gossypium hirsutum) reproductive organs, we revealed diverse roles that VIN plays in multiple reproductive processes. A set of phenotypic and genetic studies showed significant reductions of viable seeds in GhVIN1-RNAi plants, attributed to pollination failure and impaired male and female fertilities. The former was largely owing to the spatial mismatch between style and stamen and delayed pollen release from the anthers, whereas male defects came from poor pollen viability. The transgenic stamen exhibited altered expression of the genes responsible for starch metabolism and auxin and jasmonic acid signaling. Further analyses identified the reduction of GhVIN expression in the seed coat as the major cause for the reduced female fertility, which appeared to disrupt the expression of some key genes involved in trehalose and auxin metabolism and signaling, leading to programmed cell death or growth repression in the filial tissues. Together, the data provide an unprecedented example of how VIN is required to synchronize style and stamen development and the formation of male and female fertilities for seed development in a crop species, cotton.

RUNX super-enhancer control through the Notch pathway by Epstein-Barr virus transcription factors regulates B cell growth.

In B cells infected by the cancer-associated Epstein-Barr virus (EBV), RUNX3 and RUNX1 transcription is manipulated to control cell growth. The EBV-encoded EBNA2 transcription factor (TF) activates RUNX3 transcription leading to RUNX3-mediated repression of the RUNX1 promoter and the relief of RUNX1-directed growth repression. We show that EBNA2 activates RUNX3 through a specific element within a −97 kb super-enhancer in a manner dependent on the expression of the Notch DNA-binding partner RBP-J. We also reveal that the EBV TFs EBNA3B and EBNA3C contribute to RUNX3 activation in EBV-infected cells by targeting the same element. Uncovering a counter-regulatory feed-forward step, we demonstrate EBNA2 activation of a RUNX1 super-enhancer (−139 to −250 kb) that results in low-level RUNX1 expression in cells refractory to RUNX1-mediated growth inhibition. EBNA2 activation of the RUNX1 super-enhancer is also dependent on RBP-J. Consistent with the context-dependent roles of EBNA3B and EBNA3C as activators or repressors, we find that these proteins negatively regulate the RUNX1 super-enhancer, curbing EBNA2 activation. Taken together our results reveal cell-type-specific exploitation of RUNX gene super-enhancers by multiple EBV TFs via the Notch pathway to fine tune RUNX3 and RUNX1 expression and manipulate B-cell growth.

Exploiting kinase polypharmacology for nerve regeneration.

Exploiting kinase polypharmacology for nerve regeneration.

A Tumor-specific MicroRNA Recognition System Facilitates the Accurate Targeting to Tumor Cells by Magnetic Nanoparticles.

Targeted therapy for cancer is a research area of great interest, and magnetic nanoparticles (MNPs) show great potential as targeted carriers for therapeutics. One important class of cancer biomarkers is microRNAs (miRNAs), which play a significant role in tumor initiation and progression. In this study, a cascade recognition system containing multiple plasmids, including a Tet activator, a lacI repressor gene driven by the TetOn promoter, and a reporter gene repressed by the lacI repressor and influenced by multiple endogenous miRNAs, was used to recognize cells that display miRNA signals that are characteristic of cancer. For this purpose, three types of signal miRNAs with high proliferation and metastasis abilities were chosen (miR-21, miR-145, and miR-9). The response of this system to the human breast cancer MCF-7 cell line was 3.2-fold higher than that to the human breast epithelial HBL100 cell line and almost 7.5-fold higher than that to human embryonic kidney HEK293T cells. In combination with polyethyleneimine-modified MNPs, this recognition system targeted the tumor location in situ in an animal model, and an ~42% repression of tumor growth was achieved. Our study provides a new combination of magnetic nanocarrier and gene therapy based on miRNAs that are active in vivo, which has potential for use in future cancer therapies.

Green Tropical Phytoextracts - Promising Anticancer Alternative

The anticancer potential of aqueous extracts of tropical plants Ficus deltoidea and Labisia pumila were tested on human prostate carcinoma (DU145) cells. The effects of these extracts on the cells were observed through several tests such as cytotoxicity MTT assay, trypan blue exclusion assay (for cell viability), fluorescence and morphological observations. Both of the plant extracts had a growth repression effect on the cells with different concentrations. The best concentration (1x10-3 μg/mL) was chosen by determining their cytotoxic effects on human prostate cancer as well as fibroblast (normal) cells. The F. deltoidea extract showed more potency and killed the cells faster than L. pumila. Morphologically both extracts affected the cells in a similar way, i.e. the cells became irregular shaped, detached themselves and kept floating in the medium.

Growth inhibition and chemo-radiosensitization of head and neck squamous cell carcinoma (HNSCC) by survivin-siRNA lentivirus

BackgroundSurvivin expression is often associated with aggressive tumor behavior and therapy resistance. In this study, we investigated the effect of survivin knockdown by survivin-siRNA lentiviral vector (Svv-Lent) on the response of HNSCC to chemo-radiotherapy, tumor growth and metastasis. MethodsFour human HNSCC (OSC19, Cal27, Cal33 and FaDu) and one normal HOK cell lines were included in the study, and survivin knockdown was achieved with Svv-Lent treatment. Cell proliferation and apoptosis were measured by MTT and TUNEL assay, respectively. Transwell assays were performed to measure in vitro cell migration and matrigel invasion. Xenograft tumors were developed in nude mice by injecting Cal27 cells subcutaneously and following tail-vein injection of lung and liver metastasis. ResultsKnockdown of survivin significantly suppressed HNSCC cell proliferation and induced apoptosis in vitro. Survivin inhibition could also significantly reduce in vitro cell migration and matrigel invasion that might be due to inactivation of matrix metalloproteinases. In vivo studies showed significant repression of Cal27 xenograft tumor growth and tissue metastasis leading to improvement in mice survival in the Svv-Lent treated group compared to controls. Our data indicated that survivin expression in HNSCC cells contributed to chemo-radioresistance, and its down-regulation increased anti-cancer effects of paclitaxel, cisplatin and radiation. ConclusionsOur findings suggest that sustained survivin expression facilitates HNSCC tumor growth and confers resistance to chemo-radiotherapy. Svv-Lent therapy may be able to enhance the cytotoxic effect of commonly used anticancer drugs such as cisplatin and paclitaxel, and radiotherapy that could provide a promising strategy for the effective control of resistant head and neck cancer.

Actin-dependent vacuolar occupancy of the cell determines auxin-induced growth repression

The cytoskeleton is an early attribute of cellular life, and its main components are composed of conserved proteins. The actin cytoskeleton has a direct impact on the control of cell size in animal cells, but its mechanistic contribution to cellular growth in plants remains largely elusive. Here, we reveal a role of actin in regulating cell size in plants. The actin cytoskeleton shows proximity to vacuoles, and the phytohormone auxin not only controls the organization of actin filaments but also impacts vacuolar morphogenesis in an actin-dependent manner. Pharmacological and genetic interference with the actin-myosin system abolishes the effect of auxin on vacuoles and thus disrupts its negative influence on cellular growth. SEM-based 3D nanometer-resolution imaging of the vacuoles revealed that auxin controls the constriction and luminal size of the vacuole. We show that this actin-dependent mechanism controls the relative vacuolar occupancy of the cell, thus suggesting an unanticipated mechanism for cytosol homeostasis during cellular growth.

Novel Functional Roles for Ten-Eleven-Translocation 2 (Tet2) in Normal and Leukemic Growth of Mast Cells

KIT receptor signaling plays an important role in mast cell development. Gain-of-function mutations in KIT receptor have been identified in human diseases including gastrointestinal stromal tumors (GIST), systemic mastocytosis (SM) and acute myeloid leukemia (AML). Although KIT mutations found in GIST are sensitive to imatinib, KIT mutation (KITD816V) found in 90% of SM patients is imatinib-resistant and currently no therapies are available to treat the human diseases associated with this mutation. Our recent studies have identified Ten-Eleven-Translocation 2 (TET2) mutations in ~23% of SM patients and are associated with poor prognosis and overall survival. TET2 is a methylcytosine dioxygenase that plays a vital role in active DNA demethylation. Recent studies suggest that patients with mutations in TET2 and KITD816V develop more aggressive form of mastocytosis with worse prognosis. Although it is known that TET2 and KITD816V cooperate in SM patients, it is not clear how they cooperate with each other and what is the physiologic role of TET2 in normal mast cell development. We show that loss of Tet2 results in impaired maturation of mast cells in vivo and in bone marrow-derived mast cells (BMMC) compared to WT controls, which is associated with reduction in 5-hmc levels compared to WT BMMCs. We also observed reduction in the expression of mast cell-specific genesincluding Mast cell proteinase-5 (MCP-5), Mast cell proteinase-6 (MCP-6) and Carboxypeptidase A (CPA). To determine the mechanism behind altered mast cell differentiation in Tet2-/- BMMCs, we performed RNA-seq analysis in WT and Tet2-/- mast cells and observed altered expression of various genes involved in development of mast cells including Kit, FcεR1, Mitf, Notch, and Myc. We further confirmed altered expression of Mitf, Gata-2, and PU.1 in Tet2-/- BMMCs compared to WT BMMCs by western blotting. Since Tet2 regulates DNA demethylation, we tested whether altered BMMC differentiation in Tet2-/- mice is due to enhanced DNA methylation. We treated WT or Tet2-/- BM cells for 3 weeks with vehicle or 5-azacytidine (hypomethylating agent) and analyzed mast cell differentiation. Treatment with 5-azacytidine completely corrected the defective mast cell differentiation in Tet2-/- cells to WT levels. These results suggest that Tet2 plays a significant role in mast cell differentiation by regulating the expression of critical transcription factors including Mitf, Gata-2 and PU.1. We next analyzed the growth of Tet2-/- BMMCs in response to cytokines. Tet2-deficient BMMCs show enhanced cytokine mediated growth compared to WT BMMCs. Hyper-proliferation of Tet2-/- BMMCs is associated with reduced expression of tumor suppressor, PTEN, whose promoter is hypermethylated and a concomitant increase in the activation of the PI3K/AKT pathway. Since loss of function TET2 mutations have been observed in SM patients in addition to KITD816V mutation, we tested whether loss of Tet2 cooperates with KIT mutation in vitro and in vivo. Tet2-deficiency or knockdown in conjunction with the expression of KIT mutation resulted in significantly enhanced growth compared to cells bearing KIT mutation alone or lacking Tet2 expression. Likewise in human mastocytosis xenograft model, significantly enlarged tumors were observed in NSG mice transplanted with human mastocytosis cell line bearing the KITD816V mutation (HMC1.2) and knockdown of TET2 compared to HMC1.2 cells bearing only the KITD816V mutation. The cooperation between loss of Tet2 and KIT mutation was associated with further increase in PI3K/AKT activation and pharmacologic inhibitor treatment with a PI3K inhibitor GDC-0941 (Pan PI3K), but not TGX221 (p110β-specific) or IC87114 (p110δ-specific), significantly reduced the hyper-proliferation of Tet2-/- BMMCs and cell lines as well as primary BM blasts derived from SM patients bearing the KITD816V mutation. Consistently, combined loss of p110α and p110δ subunits of PI3K resulted in the most profound growth repression in oncogenic KIT bearing BM cells, but did not correct altered differentiation in Tet2-/- BMMCs. Taken together our results suggest that combinational therapy involving 5-azacytidine (which corrects the impaired mast cell differentiation) and PI3K inhibitor (which corrects the excessive proliferation) is a better therapeutic option for treating human mastocytosis patients bearing TET2 and KIT mutations. DisclosuresNo relevant conflicts of interest to declare.

Abstract C126: PRLR ADC: A novel antibody drug-conjugate for the treatment of PRLR positive breast cancer

Abstract Breast cancer is a heterogeneous disease comprised of various subtypes based on pathology and molecular profiling. Expression of hormone receptors (HR) and HER2 biomarkers are important determinants of therapy choice, due to the established role of these proteins as drivers of disease. Prolactin Receptor (PRLR) is a type 1 cytokine receptor that is expressed on a subset of breast cancers and may contribute to pathogenesis. Functionally, activation of PRLR by the hormone ligand Prolactin (PRL) induces PRLR dimerization and signaling resulting in cell proliferation and differentiation. While PRLR is expressed at low levels in some normal human tissues including the mammary gland, it is relatively overexpressed in ∼25% of human breast tumors and importantly, is rapidly internalized upon binding of anti-PRLR antibodies. We developed an anti-PRLR antibody-drug conjugate (ADC), PRLR ADC, to target PRLR positive breast cancer. PRLR ADC is comprised of a fully human high affinity function-blocking anti-PRLR IgG1 antibody conjugated via a non-cleavable SMCC linker to the cytotoxic maytansine derivative DM1. Both unconjugated anti-PRLR antibody and the PRLR ADC block PRL mediated activation in vitro and induce rapid internalization of the receptor into lysosomes. PRLR ADC induces potent cell cycle arrest and cytotoxicity in several PRLR-expressing cell lines. The in vivo efficacy of PRLR ADC was explored in breast cancer cell line xenograft models expressing both endogenous PRLR (MCF7, T47D) or transfected receptor (MCF7/PRLR). Treatment of tumor bearing SCID (T47D) or NCr Nude (MCF7) animals was initiated approximately 15 days post implantation of cells where tumor volumes averaged 150-200 mm3. In both T47D and MCF7/PRLR xenograft models, where PRLR is expressed highly, single or multiple (once weekly x 3) doses of 2.5-15 mg/kg resulted in significant inhibition of tumor xenograft growth. In the MCF7 model that expresses low levels of PRLR, inhibition and regression of tumors was observed at 10 and 15 mg/kg dose levels. In all models, higher doses resulted in greater and more prolonged repression of tumor growth. Conjugation of DM1 to anti-PRLR antibody was required for efficacy, as unconjugated antibody had no effect on tumor growth. Anti-tumor efficacy of PRLR ADC was also assessed in NSG mice bearing breast cancer Patient Derived Xenograft (PDXs) tumors with moderate and heterogeneous expression of PRLR. Treatment was initiated 21 days after implantation of the PDX tumors where the average tumor volume was ∼500mm3. Anti-tumor efficacy was observed following 10 or 20 mg/kg PRLR ADC dosed once weekly x 4. These studies demonstrate the promising anti-tumor activity of the PRLR ADC against PRLR positive breast cancers and support the continued development of this agent. Citation Format: Marcus P. Kelly, Sandra Coetzee, Carlos Hickey, Sosina Makonnen, Frank Delfino, Julian Andreev, Arthur Kunz, Christopher D'Souza, Jason Giurleo, Thomas Nittoli, Pamela A. Trail, Nicholas Papadopoulos, Gavin Thurston, Jessica R. Kirshner. PRLR ADC: A novel antibody drug-conjugate for the treatment of PRLR positive breast cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C126.

Hormonal control of cold stress responses in plants.

Cold stress responses in plants are highly sophisticated events that alter the biochemical composition of cells for protection from damage caused by low temperatures. In addition, cold stress has a profound impact on plant morphologies, causing growth repression and reduced yields. Complex signalling cascades are utilised to induce changes in cold-responsive gene expression that enable plants to withstand chilling or even freezing temperatures. These cascades are governed by the activity of plant hormones, and recent research has provided a better understanding of how cold stress responses are integrated with developmental pathways that modulate growth and initiate other events that increase cold tolerance. Information on the hormonal control of cold stress signalling is summarised to highlight the significant progress that has been made and indicate gaps that still exist in our understanding.

Inhibitory effects of fermented extract of Ophiopogon japonicas on thrombin-induced vascular smooth muscle cells.

Ophiopogon japonicus is known to have various pharmacological effects. The present study investigated the effects of an extract of fermented Ophiopogon japonicas (FEOJ) on thrombin‑treated vascular smooth muscle cells (VSMCs). FEOJ treatment inhibited the proliferation of VSMCs treated with thrombin as indicated by an MTT assay. These inhibitory effects were associated with decreased phosphorylation of AKT, reduced expression of cyclin D1 and increased expression of p27KIP1 in thrombin‑induced VSMCs. In addition, FEOJ treatment suppressed the thrombin‑stimulated migration of VSMCs as demonstrated by a wound‑healing migration assay. Furthermore, zymographic analyses demonstrated that treatment of FEOJ with VSMCs suppressed the thrombin‑induced expression of matrix metalloproteinase (MMP)‑2, which was attributed to the reduction of nuclear factor (NF)‑κB binding activity. Collectively, these results demonstrated that FEOJ induced p27KIP1 expression, reduced cyclin D1 expression and AKT phosphorylation, and inhibited MMP‑2 expression mediated by downregulation of NF‑κB binding activity in thrombin‑treated VSMCs, which led to growth inhibition and repression of migration. These results supported the use of FEOJ for the prevention of vascular diseases and provided novel insight into the underlying mechanism of action.

Divergent Androgen Receptor and Beta-Catenin Signaling in Prostate Cancer Cells.

Despite decades of effort to develop effective therapy and to identify promising new drugs, prostate cancer is lethal once it progresses to castration-resistant disease. Studies show mis-regulation of multiple pathways in castration-resistant prostate cancer (CRPC), reflecting the heterogeneity of the tumors and also hinting that targeting androgen receptor (AR) pathway alone might not be sufficient to treat CRPC. In this study, we present evidence that the Wnt/β-catenin pathway might be activated in prostate cancer cells after androgen-deprivation to promote androgen-independent growth, partly through enhanced interaction of β-catenin with TCF4. Androgen-independent prostate cancer cells were more prone to activate a Wnt-reporter, and inhibition of the Wnt/β-catenin pathway increased sensitivity of these cells to the second-generation antiandrogen, enzalutamide. Combined treatment of enzalutamide and Wnt/β-catenin inhibitor showed increased growth repression in both androgen-dependent and -independent prostate cancer cells, suggesting therapeutic potential for this approach.

Dual mTOR inhibitor MLN0128 suppresses Merkel cell carcinoma (MCC) xenograft tumor growth

Merkel cell carcinoma (MCC) is an aggressive neuroendocrine skin cancer. Pathologic activation of PI3K/mTOR pathway and elevated expression of c-Myc are frequently detected in MCC. Yet, there is no targeted therapy presently available for this lethal disease. Recently, MLN0128, a second-generation dual TORC1/2 inhibitor is shown to have therapeutic efficacy in preclinical studies. MLN0128 is currently in clinical trials as a potential therapy for advanced cancers. Here we characterize the therapeutic efficacy of MLN0128 in the preclinical setting of MCC and delineate downstream targets of mTORC1/2 in MCC cellular systems. MLN0128 significantly attenuates xenograft MCC tumor growth independent of Merkel cell polyomavirus. Moreover, MLN0128 markedly diminishes MCC cell proliferation and induces apoptosis. Further investigations indicate that senescence does not contribute to MLN0128-mediated repression of xenograft MCC tumor growth. Finally, we also observe robust antitumor effects of MLN0128 when administered as a dual therapy with JQ1, a bromodomain protein BRD4 inhibitor. These results suggest dual blockade of PI3K/mTOR pathway and c-Myc axis is effective in the control of MCC tumor growth. Our results demonstrate that MLN0128 is potent as monotherapy or as a member of combination therapy with JQ1 for advanced MCC.

S-nitrosylation triggers ABI5 degradation to promote seed germination and seedling growth.

Plant survival depends on seed germination and progression through post-germinative developmental checkpoints. These processes are controlled by the stress phytohormone abscisic acid (ABA). ABA regulates the basic leucine zipper transcriptional factor ABI5, a central hub of growth repression, while the reactive nitrogen molecule nitric oxide (NO) counteracts ABA during seed germination. However, the molecular mechanisms by which seeds sense more favourable conditions and start germinating have remained elusive. Here we show that ABI5 promotes growth via NO, and that ABI5 accumulation is altered in genetic backgrounds with impaired NO homeostasis. S-nitrosylation of ABI5 at cysteine-153 facilitates its degradation through CULLIN4-based and KEEP ON GOING E3 ligases, and promotes seed germination. Conversely, mutation of ABI5 at cysteine-153 deregulates protein stability and inhibition of seed germination by NO depletion. These findings suggest an inverse molecular link between NO and ABA hormone signalling through distinct posttranslational modifications of ABI5 during early seedling development.

Manipulation of cold stratification and endophytic effects on expression patterns of RSG and KAO genes in coleorhiza of wheat seeds

Stratification is the exposure of seeds to conditions which break dormancy and enhance germination. Currently stratification is limited to abiotic conditions such as cold, acidity, light, salinity and osmotic priming. Here, the role of biological stratification on mycovitality was uncovered by assessing the effect of fungal endosymbiont on its host plant during dormancy release and germination commencement in seeds. The impact of the endophytic fungal SMCD 2206 strain on germination energy and vigor as compared to untreated seeds was evaluated. The expression patterns of plant growth promoting genes, repression of shoot growth (RSG) protein and ent-kaurenoic acid oxidase (KAO) of gibberellins (GAs), during the early breakage of seed dormancy and germination were assessed and indicated the involvement of a GA-mediated negative feedback regulation in treated seeds. The GA levels in all treated seeds reached optimum before 24 h of germination with direct interactions between seeds and endophyte generating maximum GA levels during the early germination stage. Our findings bring a significant contribution to plant prenatal care of cereal crops; they demonstrate endophyte-mediated vitality, a reprogramming of pre- and post-germination events in wheat seeds, which enhances dormancy breakage and germination.

Elevated guanosine 5'-diphosphate 3'-diphosphate level inhibits bacterial growth and interferes with FtsZ assembly.

FtsZ, a protein essential for prokaryotic cell division, forms a ring structure known as the Z-ring at the division site. FtsZ has a GTP binding site and is assembled into linear structures in a GTP-dependent manner in vitro. We assessed whether guanosine 5'-diphosphate 3'-diphosphate (ppGpp), a global regulator of gene expression in starved bacteria, affects cell division in Salmonella Paratyphi A. Elevation of intracellular ppGpp levels by using the relA expression vector induced repression of bacterial growth and incorrect FtsZ assembly. We found that FtsZ forms helical structures in the presence of ppGpp by using the GTP binding site; however, ppGpp levels required to form helical structures were at least 20-fold higher than the required GTP levels in vitro. Furthermore, once formed, helical structures did not change to the straight form even after GTP addition. Our data indicate that elevation of the ppGpp level leads to inhibition of bacterial growth and interferes with FtsZ assembly.

Abstract CN05-03: Cooperation between a rexinoid and EGFR-TKI for lung cancer prevention via Cyclin D1 destabilization

Abstract We previously reported that both nuclear retinoic acid receptor (RAR, retinoid) and retinoid X receptor (RXR, rexinoid) agonists can trigger proteasomal degradation of cyclins. This confers check point arrest and repair of carcinogenic DNA damage in bronchial epithelial cells. Mechanisms responsible for this induced degradation were discovered. These included ubiquitin-dependent as well as ISG15-dependent programs that independently destabilized expression of cyclin D1 and other G1 cyclin proteins. The critical receptor that confers this cyclin destabilization was RARβ. Yet, silencing of RARβ; and specifically of the previously unrecognized isoform that we cloned and designated as RARβ1 likely accounts for clinical resistance to classical retinoids (like 13-cis-retinoic acid and all-trans-retinoic acid) in lung carcinogenesis. We sought to learn whether RXR/RAR heterodimer complex activation with a rexinoid was able to trigger cyclin destabilization. This was found to be the case in in vitro studies. This finding implied that the same pathway would be engaged in the in vivo setting. To establish if this occurs, we engineered transgenic mice independently with human surfactant C-driven wild-type cyclin E or a proteasome-degradation resistant cyclin E species. This mouse model was developed because human pre-malignant and malignant lung lesions frequently deregulate cyclin expression. Intriguingly, these mice recapitulated many features of lung carcinogenesis found in patients. Neoplastic changes were enhanced by transgenic expression of the degradation-resistant cyclin E species. We built on this finding by showing that a rexinoid and an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) each chemoprevented lung cancers in vinyl carbamate-induced lung tumors within strain A/J mice. Notably, the rexinoid was more potent that the EGFR-TKI in conferring this chemoprevention. Both agents also reduced cyclin expression, but did so through distinct mechanisms. We sought to learn if a rexinoid (bexarotene) cooperated with an EGFR-TKI (erlotinib) in exerting anti-tumorigenic effects in lung cancer. Cooperation between these agents was found in both murine and human lung cancer cells and even in those cells that exhibited KRAS/p53 mutations. Over the past decade, we translated this work into the cancer clinic through a series of five clinical trials that moved this work through successive phase 0, phase I, and phase II trials of lung and aerodigestive tract cancers. In window of opportunity trials, pharmacodynamic responses (cyclin repression and induced necrosis and inflammatory responses) were seen when intratumoral levels of these drugs were comparable to those necessary to trigger in vitro effects. A phase II trial in heavily pre-treated stage IV non-small cell lung cancer (NSCLC) cases was performed. EGFR and cyclin expression profiles as well as KRAS mutations were searched for in these NSCLC cases. Findings revealed substantial cyclin repression and reduction of lung cancer growth by combined therapy with a rexinoid (bexarotene) and EGFR-TKI (erlotinib). Cyclins were repressed while necrosis and inflammation were induced in post-treatment versus pre-treatment lung tumor biopsies obtained in this window of opportunity trial. Objective anti-tumor responses occurred whether or not KRAS or activating EGFR mutations were detected. This refractory NSCLC trial had 3 major clinical responses (2 had KRAS or EGFR mutations) with prolonged survival (583, 665, and 1460+ days, respectively). Median survival was 22 weeks (16 weeks for controls). Hypertriglyceridemia or rash significantly increased median overall survival to 24 weeks. Thus, this combination regimen revealed substantial clinical anti-tumor activity against NSCLCs. Taken together, these findings indicate that cooperation between a rexinoid and EGFR-TKI can chemoprevent and treat lung cancers by causing cyclin destabilization. These agents are also useful tools to identify other antineoplastics that repress lung cancers by destabilizing cyclin expression or by inhibiting their associated kinases. Evidence for this will be presented in this session. Citation Format: Konstantin H. Dragnev, Vincent Memoli, Sarah J. Freemantle, Samuel Waxman, Ethan Dmitrovsky. Cooperation between a rexinoid and EGFR-TKI for lung cancer prevention via Cyclin D1 destabilization. [abstract]. In: Proceedings of the Thirteenth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2014 Sep 27-Oct 1; New Orleans, LA. Philadelphia (PA): AACR; Can Prev Res 2015;8(10 Suppl): Abstract nr CN05-03.