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    Universidade do Algarve

    Departamento de Cincias Biomdicas e Medicina

    Mestrado Integrado em Medicina

    Mdulo de Escolha do Estudante

    Characterization of FOXO3a-mediated gene expression

    following NPV-BEZ235 exposure: A novel role for

    TRIB2

    Laboratrio Wolfgang Link

    Tutor: Richard Hill

    Realizado por: Teresa Martins (a39062)

    N de Palavras: 4311

  • Page 2 of 15

    Characterization of FOXO3a-mediated gene expression following NPV-BEZ235 exposure: A novel

    role for TRIB2

    Abstract

    Melanoma is an extremely aggressive cancer and concomitant to this aggressiveness, patient

    prognosis is poor. As a result, novel therapies and cellular targets are desperately needed. At present the

    chemical compound BEZ235 has demonstrated significant potential as an anti-cancer agent. BEZ235 is a

    potent inhibitor of PI3Ks that are constitutively active in many cancers, including melanoma. This

    deregulation results in the inactivation of the FOXO family of transcription factors, critical regulators of

    the cell cycle and apoptosis. TRIB2, a gene that has been reported to be up-regulated in some cancers, has

    also been implicated in the negative regulation of the FOXO signaling cascade, specifically the negative

    regulation of FOXO3a. Consequently TRIB2 has been implicated in melanoma resistance to various

    chemotherapeutics, including some PI3K inhibitors. Here we investigate how TRIB2 mediates PI3K

    inhibitor resistance and the role(s) of FOXO3a in this response. Our finding implicate TRIB2 influencing

    apoptosis (although not the cell cycle) and that this occurs at the level of transcription. Our findings also

    indicate that the over expression of TRIB2 deregulates the cellular balance between p53 and MDM2.

    Key words

    Cancer, Melanoma, TRIB2, FOXO3a, p53, transcription.

  • Page 3 of 15

    Introduction.

    Cancer is the uncontrolled growth of cells in the body. Among the many types of cancers that can

    rise in an organism, melanoma, originated in anomalous melanocytes, is considered one of the most

    deadly forms of cancer due to the extreme aggressiveness and significant resistance to current

    therapeutics1, 2

    . According to the World Health Organization, melanoma kills approximately 53,000

    patients per year, worldwide. Of all cancers, melanoma is considered straightforward to diagnose due to

    the presence of the melanin pigment that can be directly observed. Radiation therapy, surgical resection

    and systemic therapy (interferon, dacarbazine or others), are some of the techniques used in the treatment

    of melanoma3-5

    . Yet, since it is a disease that spreads quickly to surrounding tissues standard treatments

    do not significantly increase patient survival. Aside from surgical resection, investigators still lack to find

    a therapeutic modality that can enhance the likelihood of a curative outcome.

    Over the last few years research has yielded important breakthroughs in our understanding of

    melanoma particularly the molecular basis of the disease, the deregulated cellular processes essential for

    continued cell growth, the metastatic process and mechanisms of melanoma resistance to

    chemotherapeutics2-4, 6-10

    . However to date the only two co-adjuvant treatments approved by the United

    States of America Food and Drug Administration are high-dose Interleukin-2 and dacabarzin. NPV-

    BEZ235 (BEZ) is an imidazoquinoline that targets the phosphatedylinositol 3 kinase (PI3K) and the

    mammalian target of rapamycin (mTOR), that has demonstrated significant potential antineoplastic

    properties and is currently being tested I various active clinical trials11-15

    .

    PI3Ks (the cellular target of the chemotherapeutic BEZ) are lipid kinases that play a central role in

    the regulation of the cell cycle, apoptosis, DNA repair, senescence (terminal cell cycle arrest),

    angiogenesis, cellular metabolism, and motility16

    . The deregulation that leads to diminished cell death and

    increased growth and proliferation is driven by the accumulation of genetic and epigenetic alterations

    within the cancer. The activation of PI3Ks has a noticeable relation with tumor growth, since it promotes

    an increase in cellular mass, cell cycle entry, counteracts apoptosis, modulates and controls cytoskeletal

    rearrangements and enhances cell migration and metastasis. PI3K activation leads to the production of

    phosphatidylinositol 3,4,5-triphosphate, that activates Phosphoinositide-dependent kinase-1 (PDK1) and

    Protein Kinase B (Akt)17, 18

    . The Akt kinase promotes cell survival by phosphorylating, and thereby

    inactivating, pro-apoptotic factors, such as the FOXO transcription factor family. The FOXO proteins

    (including FOXO3a) play an important role in longevity and tumor suppression by regulating a wide

    range of genes involved in stress resistance, metabolism, cell cycle arrest and apoptosis19-21

    .

    Previous studies have shown that BEZ treatment of malignant melanoma cells induces FOXO3a-

    dependent gene expression following the inhibition of PI3K1, 14

    . Functional studies suggest that the tumor

    suppressive role of was due to their inactivation, caused by constitutively active PI3K/Akt or Ras/

    mitogen-activated protein kinase (MAPK)/ERK signaling. Activation of these pathways can directly

  • Page 4 of 15

    result in phosphorylation of FOXOs and their subsequent cytoplasmic sequestration and/or degradation

    via the ubiquitin-proteasome pathway. When FOXO is activated by the inhibition of the PI3K/Akt

    pathway, FOXOs promotes a wide range of effects including cell cycle arrest, cell differentiation,

    autophagy and apoptosis via various mechanisms22

    .

    Tribbles-2 (TRIB2) is a kinase-like protein with a role in cell division. Further TRIB2 has been

    reported to be up-regulated in a subset of acute myeloid leukaemias. Members of tribbles family have

    been reported to interact and modulate the activity of signal transduction pathways, including the

    PI3K/Akt and the MAPK systems. Furthermore, TRIB2 has been implicated in the negative regulation of

    FOXO3a23, 24

    . The FOXO family of proteins that are transcription factors regulating cell cycle arrest (for

    example the cyclin-dependent kinase inhibitor 1B p27) and pro-apoptotic (e.g. Bcl-2 interacting mediator

    of cell death Bim) gene transcription22

    . The restoration of FOXO proteins have been suggested as a

    promising strategy to treat various types of cancer and accordingly, the forced expression of nuclear

    FOXO has been shown to induce apoptosis in a wide range of in vitro cancer cell line models24

    . TRIB2

    (that is highly expressed in metastatic melanoma cells) has been implicated in the resistance of various

    cancers to a range of chemotherapeutics, including PI3K inhibitors that are under clinical trial. It is

    hypothesized that this resistance is due to the repression of FOXO family members21

    .

    Based on my groups previous and current research, my project was to elucidate some of the

    mechanism(s) of action regarding how TRIB2 mediates PI3K inhibitor resistance and the role(s) of

    FOXO3a in this response. To test this hypothesis I confirmed the inhibition effectiveness of our PI3K

    inhibitor, confirmed the cellular phenotype following TRIB2 over expression and examined the

    recruitment of FOXO3a the promoters of key cell cycle arrest or pro-apoptotic gene promoters.

    Methods.

    Tissue Culture

    The cell lines used for this project were G631 (human melanoma) and the U2OS (human

    Osteosarcoma). The cells were cultivated in 15 cm plates with 10% heat inactivated FCS (Sigma)

    supplemented with Pen/Strep (Gibco). The U2OS cell line was previously transfected with a plasmid

    containing the TRIB2. The G361 cell line was stably transfected with a TRIB2 shRNA expressing

    plasmid. BEZ239 was used at 100 nM for all studies.

    Imunnoblotting/Western Blotting

    Total protein was extracted from each cell line For cellular extraction, the cells were collected from

    the culture plates by first removing the growth medium and to then scrape the cells in 1 ml. This

    suspension was then transferred into a clean Eppendorf and spinned at 1100 rpm for 5 min at 4C. The

    PBS was removed from the pellet and RIPA buffer (Tris-HCL ph 7.4, NaCl, 10% Nonidet P-40, 10%

    sodium deoxycholate, 100 mM EDTA, PIC, 200 mM NA-F, 100mM Na3VO4 and protease inhibitors

    cocktail) was added to the pellet. The pellet was resuspended in this total lysis buffer and incubated on ice

  • Page 5 of 15

    for 30 minutes. After 30 minutes the lysed cells were spun 15 minutes at 13000 rpm (maximum speed).

    The supernatant (containing our proteins) was collected and transferred to a fresh eppendorf prior to

    quantification. All extracted proteins were stored at -80OC until required.

    To determine the protein concentrations in each sample we used the Quick StratTM Bradford

    Protein Assay (BioRad) and the NanoDrop 2000 UV-Vis Spectrophotometer (ThermoScientific)

    following the manufacturers guidelines.

    Our extracted protein samples protein were diluted in to 2x lammeli loading buffer (containing -

    mercaptoethanol) and heatedat 95C for 5 minutes. Samples were loaded into our 10% SDS-PAGE gels.

    Separated proteins within each gel were transferred on to nitrocellulose membranes (A