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Immunoprecipitation

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  • Keith:Thank you Michael Fiske for the wonderful introduc. My collegues and I are here, at the diseased genome hospital, to discuss a very prominent disease: LUNG CANCER.
  • Please follow us into examination room J200. Please Have a seat.
  • Let me pull up ur chart real quick.I first want to explain to you what’s going on. Here’s an xray of a healthy set of lungs. You can tell they’re healthy because they are clear.
  • I asked you to come in today, because I wanted to discuss something that is very concerning to us. Here is your chest xray from your last visit. What concerns us is this: CLICK. ARROW. There appears to be a mass of some sort, on your left lung.The symptoms you’ve previously described during your last visitCoughingwheezingshortness of breathchest paindifficulty swallowinghoarsenessloss of appetiterespiratory infection This has led my colleduges and I to speculate you have LCBefore you come too upset, let me col. And I explain to you where we’re going to go with this apt today.
  • Today we will be discussing the different parts that could be wrong with ur body and could be causing this lc. We are hoping that this will provide you with a clear understanding of what you may be giong through and the future possiblities.First, we’ll start off today talking about what is lung cancer, the types there are the type that you may have. Also, the risks that led you to this disease. Then we r gonna talk about some mechs that could be going wrong in ur body. There are some poss genetic predis that could lead u to lc. Then we will talk about various mutations..Angiogenesis is key to what you may have, DO YOU KNOW WHAT THAT IS?Dr. Ross will take it from here.
  • Liz:Cancer starts out as a group of cells that are dividing continuisly. As the cancer progresses, cells have divided so frequently that they’ve begun to form solid masses known as tumors. That can be seen in ur pamphlet. Cells proliferate and invade other regions of tissue that theyre in. eventually these cells grow so much that they enter the blood suppy where they metastasize to other regions of the body. Right now u have a tumor in ur lung that has not yet metastasized but we should treat it beforeWe’ll give you a roadmap about your disease…after this slideLc background10 min introLiz: egfr mutantsangiogenesis: elliotKeith: EGFR +other proteinsBobby: chromosomal lociNatalie: cisplatin treatmentDraw tumorepithelial cell with blood vessels coming in, nucleus containing chromosomes,efgr receptor + other proteins, met, vegf, megf, drugs on outside, killing cell End intro. 5 main parts of lc that we’ll be talking about today. Parts that involve the chromosome (highlight with box) this what bobby will talk to u about today. Theres also mutations on the egfr. In addition to egfrtheres these proteins that well be talking about. How tumor cells get drug supply. And how drugs kill tumor cells?Each time u end a section show this image again and have summarizing wordsDim old parts and highlight new parts
  • The distribution of cigarettes first began in World War II (1938-1944) to soldiers. In the 1940s and early '50s, cigarette smoking became more commonplace due to advertising. Cigarette ads usually featured people of a higher class or soldiers and groups laughing and having fun. People were also permitted to smoke anywhere they wanted, including nurse stations in hospitals, salons, barbershops, cafes and the grocery store. Lung cancer was first described by doctors in the mid-19th century. In the early 20th century it was considered relatively rare, but by the end of the century it was the leading cause of cancer-related death among men in more than 25 developed countries. In the United States it has surpassed breast cancer as the leading cause of death from cancer among women. This rapid increase is due mostly to the increased use of cigarettes since World War I.
  • 2 flavors: nclc and small cell.Nsc is what you have. Thankfully u have nsclc which is more treatable.Liz:Even the two main forms of lung cancer are diverse. There are three types of NSCLC: squamous cell carcinoma originates near the bronchus and which affects the squamos epithelial cells, adenocarcinoma which originates on the outer region of the lungs, & large-cell carcinoma which can originate at any part of the lung. NSCLC is classified by the cells in the different subtypes (either by size, shape, or chemical make up). Small cell lung cancer grows and spreads much more rapidly than NSCLC. This type of cancer is characterized by tiny cells that are referred to as OAT cells. These can be seen in the image on the right. So these are the cancers, but lets take a look at what causes the chaos at the cell and molecular level of nsclc. The development of lung cancer is not the result of a sudden, transforming event in the bronchial epithelium. Rather, it is a multi-step process involving sequential genetic and cellular changes, predominantly in recessive oncogenes
  • Eliot:TreatmentsSurgery, that’s very abrasive and very riskyRadiation-ChemotherapyVarious Drugs
  • This picture might seem very overwhelming, but what I want you to take away fro mthis is that there aer many poss in ur body that may be causing urlc and my collegeues and I will be discussing these various poss.Dr. Hodges will be discussing the genetic predispositionsDr. Ross will be talking about mutations in EGFR protein kinaserecep.Dr. Solvang will be talking further about RGFR and ERGG2, as well as other mutated proteins that associate with lcDr. Vildaver will be meeting with u to discuss VEGF and its role in angiogenesis Finally, Dr. Simak present to u with various treatment options and find the proper course of treatment.
  • Big ques before thisAre there regions of the chromosomes that might predis u to get lcEvidence y looking at this (slide before this)Main topic :CL- prev evidence ques (methods)resultssignificancemodel
  • Talk more about pic2 indiv from same species but regions where single nuc are changed (snp)Used for a wide variety of things what were gonna be talking about is how snps are related to ur susceptibility to lc
  • The way that they use snps to look at predis. To disease is called genome wide…Take entire genome sequence it and looks for snps, where they are and what genesetheyre in and what role geneses are playing in the diseaseSnps that normal person doesn’t have maybe snps that are key to tlcBoth might have one snps but not another
  • CITEgood
  • Not made just for nicotine. FIGURE OUTFor aceto…but nicotine can bind to nar receptors
  • Explain betterMethods?Looking at ppl from diff regions
  • Ppl who have a certain geneare more likely to smokeIs it a snp?
  • How related to lc
  • This picture might seem very overwhelming, but what I want you to take away fro mthis is that there aer many poss in ur body that may be causing urlc and my collegeues and I will be discussing these various poss.Dr. Hodges will be discussing the genetic predispositionsDr. Ross will be talking about mutations in EGFR protein kinaserecep.Dr. Solvang will be talking further about RGFR and ERGG2, as well as other mutated proteins that associate with lcDr. Vildaver will be meeting with u to discuss VEGF and its role in angiogenesis Finally, Dr. Simak present to u with various treatment options and find the proper course of treatment.
  • QUES: Are you aware of what the func of epidermal growth fac is?
  • First, a ligand (EGF) must bind to a receptor EGFR. A shape change occurs and EGFR is phos./activ.It then forms a heterodimer with another receptor named ERBB2. When this heterodimerization happens, they are both phos. Which provokes signalling cascade of spec. proteins which leads to the nuc.Here is an example of one specific pathway that leads to diff kinds of signaling.
  • EGFR can become mutated and when this happens, it has higher lvels of phos. Which means that the signallingcacscade is constantly turned on. you can imagine a light switch here that is constantly turned on.
  • There are many types of mutations that can occur on EGFR. Researchers have found that depending on where the EGFR mutation is located, there are diff phos. Found.Not one indiv. Tyr that must be affectedVector- Cell with the missing gene for EGFR…why no phosphorylationEGFR- Cells expressing normal amount of EGFR _no mutationAnti-Myc- the tag for EGFR- shows that it is present in the cellsRest are mutated EGFR cells. Whats important to know is that each of these mutated cell lines have a mutation at a different location along the EGFR receptorThe cells were starved of EGF (the ligand) can be thought of as “food” for 12 hrs.Then they EGF was added (10ng/ml). The phosphorylations were observed at various amount of time after the EGF was added. It is expected to see stronger phos levels at 10 mins. Whats interesting is distinct patterns of try phos among the different mutated cell lines. This is a lottt to look at so I want to focus on just a few parts. ChenIn response to EGF simulationuse a picBottom?Kinase domain with mutations but where not in kinase domain can be mut
  • There are many drugs that target TK by inhibiting the kinase domains.Treatmenthttp://www.nature.com/nrc/journal/v6/n9/images/nrc1913-f4.jpg
  • TreatmentChenShow one or twoThen clickkkkkkkkkkkk al these other mutations, don’t get signal transductionPut box around where theres no more phos. Left is control!!!As u move to right conc increasingNow lets look at all the other strains….similar storyWhat is activity??? IC50 micromolar? Activity of pathway?Myoegfr, measure of egfr protein present
  • TreatmentPedersenAn experiment was performed investigating the levels of EGFR/EGFRvIII and ERK phosphorylation in cells mock treated or treated with .01 or 2mM of gef and at the same time stimulated with 10nM EGF ofr 10 mins, 24, 48, 72 hours.Try1173 was chosen because to was found to be the most durable of the phosphorylation sites. Gefrapidlly and in a dose-dependent manner inhibits EGFR and ERK phosphorylations up to 72 h after EGF stimulation in both the high- and low-EGFR-expressing cell lines (A and B)Athe decrease in EGFR levels, in the absence of gef, in the low_EGFR-expressing cell line is due to receptor downregulation inducted by EGR, a mechanism that appears not to be functioning in the NR6W and NR6M lines.A concentration of .01 um gef seems to increase EGFRvIIIphosphorylation on residue 1173 and ERK phosphorylation after 48 and 72h of treatment (c). Could explain why gef in low concentrations induce cell proliferation and anchorage-independent growth. However 2um of gef effectively inhibits both EGFRvIII and ERK phosph in the NR6M cell line for up to 72h. The high concen of gef seems to induce degradation of both EGFR and EGFRvIII independent of EGF. This is particulary evident in the low-EGFR expressing cell line where .01 um gef inhibit EGF-mediated downregulation, resumably due to its inhibtion of the Try1045 site, but fails to do so at a concentration of 2um (A).Delete?Stress the idea that no gef. Phos.As morve to the left increase phos.
  • Log to get the curve?TreatmentPedersenHere we have prolif of cells dep on conc of gefSee that as u treat cells with gef their prolif rate decreasesBlack line-parental cell line…not mutated?Doesn’t work for all types of cancer…
  • Refer to phos levels in mutantsChenHowever, what is important to remember is that EGFR’s have many different mutations. The different mutations located in among various parts of the receptor were found to react differently to gefitinib. This is why Gef works for some and not others. In different EGFR variatn-expressing 32D cells, only cells harboring L858R, E746-A750 deletion, and G719S mutants were clearly more sensitive to gef than wild-type EGFR expressing cells. Cells expressing the L861Q mutant were also more sensitive, but to a less extent. Cells expressing S768I and E709G mutant were actually more resistant to gef than cells expressing the wild-type receptor. Interestingly, S768I and E709G were amont the mutants that lacked ubiquitination an dalso had sustained Try phosphorylation in response to EGF stimulation. It is unclear whether these properties are related to the relative resistance to gef in cells harboring these two mutants. Currently, unable to correlate particular mutations other than L858R and deletion mutants to treatment responses to gef due to the scarcity of those mutations in reported cases. How phos f mutants affects gefactivUsed 32D cells for the assayFound that 32D cells expressing L858R, E746-A750 Deletion and G719s mutants were most sensitive to gefitinib. They were not viable at the lowest does (.05) of the inhibitor after 3 days. L861Q were more sensitive to gefitinib that then 32D cells harboring wild-type EGFR but to a lesser extentS7681 and E709G were slightly more resistant to gef than wild typeInterestingly and for unknown reasons, at .05 had reproducible growth promoting ability in 32D cells expressing wild type, S7681 or E709G
  • This picture might seem very overwhelming, but what I want you to take away fro mthis is that there aer many poss in ur body that may be causing urlc and my collegeues and I will be discussing these various poss.Dr. Hodges will be discussing the genetic predispositionsDr. Ross will be talking about mutations in EGFR protein kinaserecep.Dr. Solvang will be talking further about RGFR and ERGG2, as well as other mutated proteins that associate with lcDr. Vildaver will be meeting with u to discuss VEGF and its role in angiogenesis Finally, Dr. Simak present to u with various treatment options and find the proper course of treatment.
  • You might be asking urself..
  • But before we go on, I just wanted to make sure that you Show protein as beads on a stringLasrttWhich one can be phos?Yes! The phos of these can happenImp to keep in mind
  • Protein cascade to the rightChange in gene expression
  • Show old evidence that says these are typ found in lcPic of lcMutations in 2 genesThis is y we wanna look at them
  • Even though its not the biggest contrib…
  • Green are proteins involved in cascadeAs increase conc activation of sig molec is increasingAdd textThese are the sig molc interacting withegfrrecep
  • On Average, EGFR only binds around 7.2 proteins, but increases to about 50 proteins…same for ErbB2.Important that all of these proteins that are recruited were found to have that SH2 or PTB binding domain.
  • Label y axis
  • This picture might seem very overwhelming, but what I want you to take away fro mthis is that there aer many poss in ur body that may be causing urlc and my collegeues and I will be discussing these various poss.Dr. Hodges will be discussing the genetic predispositionsDr. Ross will be talking about mutations in EGFR protein kinaserecep.Dr. Solvang will be talking further about RGFR and ERGG2, as well as other mutated proteins that associate with lcDr. Vildaver will be meeting with u to discuss VEGF and its role in angiogenesis Finally, Dr. Simak present to u with various treatment options and find the proper course of treatment.
  • Tumor cells’ vasculature is induced by continuous secretion of growth factorprotein (VEGF) affecting activity on nearby endothelial cells. In response to hypoxia, or deprivation of oxygen, malignant tumors turn on the “angiogenic switch” which tells them to secrete VEGF which binds to VEGF receptors on nearby blood vessels. The angiogenic switch causes rapid tumor growth and metastasis. Another protein that plays a role in angiogenesis is PDGF.
  • Normal angiogenesis is highly regulated, but this process is highly unregulated in tumor cells, especially in the case of NSCLC. Endothelial cells produce VEGF., which undergoes an autocrine signaling pathway in which a cell secretes a hormone or chemical messenger that binds to receptors on the same cell. VEGF is a potent proangiogenic protein that activates VEGF receptor (VEGFR) acts through receptor tyrosine kinase VEGFR-2 in angiogenesis. VEGF-A stimulates VEGFR-2 which is in high levels to promote angiogenesis, which in turn is required for metastasis of tumor cells. p-PDGFR-β Plays a role as well by acting on pericytes covering blood vessels, which secrete Ang-1 that contributes to blood vessel maturation and stability.
  • PI3-kinase is activated/phosphorylated by the majority of Receptor Tyrosine Kinases, including VEGFR-2. Phosphorylation of membrane-bound proteins can lead to the activation of these proteins’ kinase function, which sends a signal through the consequent proteins by phosphorylation. Specifically, these proteins are phosphorylated at tyrosine residues. This signal can then be sent through receptor protein/kinases such as PI3K. Activated PI3-kinase induces the phosphorylated of PIP2 to PIP3. The interaction of PIP3 with the PH domain of AKT likely induces conformational changes in AKT, thereby exposing the two main phosphorylation sites at T308 and S473. T308 and S473 phosphorylation by protein serine/threonine kinase 3'-phosphoinositide-dependent kinases 1 and 2 (PDK1 and PDK2) is required for maximal AKT activation. Activated AKT translocates to the nucleus and mediates the activation and inhibition of various targets resulting in cellular survival and cell growth and proliferation. PTEN is a specific PTPase that has no effect in the PI3K/Akt pathway in lung cancer. In total, all the proteins in the PI3K/Akt pathway must be phosphorylated to continue the signal. AKT is heavily implicated in angiogenesis.
  • Akt is a serine/threoninekinase. WhenAkt is phosphorylated, it inhibits the phosphorylation of certain proteins such as BAD, which have a role in apoptosis. Thus, apoptosis is inhibited and tumor cells continue to grow, especially with the activation of mTOR. P53 is related to cell-cycle arrest. Akt allows the phosphorylation of MDM2 to occur, which inhibits production of p53, resulting in cell cycle arrest being inhibited.
  • Ques: what is an oncogene? (Understand what it is precisely!!!)FLJ10504 is a protein that may function as an oncogene in the development of tumors. It is required for VEGF-A dependent signaling in the PI3K pathway. High levels of FLJ10540 help induce Aktphosphorylation and form a complex with PI3K. FLJ10540 is overexpressed in lung cancer tissue, and is in a positive correlation with VEGF-A overexpression. This leads to hyperactivation of the PI3K pathway. There are migratory and invasive qualities related to FLJ10540.
  • VEGF-A has a positive correlation with FLJ10540 in lung cancer cells. Stimulation with VEGF-A increased FLJ10540 protein expression, indicating that FLJ10540 has a role in enhancing complex formation with PI3K under VEGF-A stimulation. FLJ10540 overexpression associates with enhanced metastatic potential. CL1-0 cells are cancer cells. SU5416 is a VEGFR-2 tyrosine kinase inhibitor. Reduces expression of FLJ10540. FLJ10540 only shares expression patterns with VEGF-A, not VEGF-B.
  • Cancer cells exhibit more migratory and invasive abilities in those stimulated with VEGF-A alone than VEGF-A with SU5416 (Semaxinib). This effect was paralleled increase in FLJ10540 expression. Finally, the activation of PI3K/Akt is critical for FLJ10540-induced lung cancer cell migration and invasion upon VEGF-A stimulation. FLJ10540 could serve as a critical mediator of VEGF-A dependent activation of PI3K/Akt.
  • It has been observed that upon VEGF-A stimulation, increased levels of FLJ10540 can enhance AKT phosphorylation and that FLJ10540 can form a complex with PI3K. Increased levels of FLJ10540 can reinforce the association with p-110 and p-85. FLJ10540 stabilizes the PI3K complex and contributes to migration, invasion, and effects of angiogenesis prompted by VEGF-A stimulation in tumor cells.
  • IFP is marked by a reduced delivery of anticancer drugs. High levels of IFP are indicative of malignant tumors. Hypoxia is the deprivation of oxygen to parts of tumor cells that causes inability of chemotherapy and radiation effects to cure tumors. It also influences the activation of angiogenesis in tumors. Antitumor therapy cannot penetrate far enough into the tumor to destroy it and they only target rapidly-dividing cells. Tumour angiogenesis results in tortuous, leaky, and nonfunctional tumour vasculature, which contributes to tumour growth and metastasis.
  • It has been noted that PDGFR-B plays a crucial role in smooth muscle cell and pericyte recruitment. Involved in angiogenesis.
  • More recent data have suggested that abnormal vessel morphology, rather than vessel density, may be a better indicator of tumor angiogenesis.
  • Imatinib reduces tumor angiogenesis, presumably through simultaneous p-PDGFR-B inhibition and VEGF downregulation. Imatinib is an orally administered drug that disrupts the kinase activity of both p-PDGFR-B and VEGFR by binding to their respective ATP-binding sites on the protein.
  • This picture might seem very overwhelming, but what I want you to take away fro mthis is that there aer many poss in ur body that may be causing urlc and my collegeues and I will be discussing these various poss.Dr. Hodges will be discussing the genetic predispositionsDr. Ross will be talking about mutations in EGFR protein kinaserecep.Dr. Solvang will be talking further about RGFR and ERGG2, as well as other mutated proteins that associate with lcDr. Vildaver will be meeting with u to discuss VEGF and its role in angiogenesis Finally, Dr. Simak present to u with various treatment options and find the proper course of treatment.
  • eliot
  • K
  • K

Immunoprecipitation Immunoprecipitation Presentation Transcript

  • Lung Cancer: The Breathtaking Battle of TKIs and EGFR Mutants
    Bobby Hodges, Elizabeth Ross, Natalie Simak, Keith Solvang and Eliot Vildaver
  • Diseased
    Genome
    Hospital
    http://blogs.usask.ca/medical_education/archive/ElksRehabFront%2520Door.jpg
  • http://1.bp.blogspot.com/_HI2mCx5P3H0/SLlEQoHj8AI/AAAAAAAAA8Q/My7tf78TNc8/s320/hospital-bed-786282.jpg
  • http://img.quamut.com/chart/9471/02_healthy_sick_x-ray.jpg
  • http://www.cardiothoracicsurgery.org/content/2/1/16/figure/F1?highres=y
  • Where Are We Headed?
    http://www.freefoto.com/images/21/16/21_16_2---Single-Carriageway-Road_web.jpg
  • Cancer
    http://www.odec.ca/projects/2005/thog5n0/public_html/Tumour.gif
  • History of Lung Cancer
    http://www.icowman.com/uploadfiles/2007072409225175.jpg
    http://www.icowman.com/uploadfiles/2007072409225175.jpg
  • Pathology
    Non Small Cell
    Squamous cell carcinoma
    Adenocarcinoma
    Large-cell carcinoma
    Small Cell
    Bronchi
    Grows rapidly
    Spreads quickly
    http://images.google.com/imgres?imgurl=http://upload.wikimedia.org/
    Lynch et al., (2004)
    http://images.google.com/imgres?imgurl=http://www.dkimages.com/discover/previews/852/20223038.JPG
  • Risk Factors
    http://www.taconichills.k12.ny.us/webquests/noncomdisease/lungcancerpic.jpg
  • You Are Not Alone
    This year approximately 200,000 people will be diagnosed with lung cancer
    NSCLC accounts for 80% of all lung cancers
    http://www.cdc.gov/cancer/lung/statistics/
    http://seo2.0.onreact.com/wp-content/uploads/2008/09/you-re-not-alone.jpg
  • Epidemiology
    http://medicineworld.org/cancer/lung/
    http://images.google.com/
  • What Next?
    While there is no known cure as of right now, there ARE options!
    Imatinib
    Gefinitib
    Cisplatin
    http://dspace.mit.edu
    http://dspace.mit.edu
  • EGF
    ERBB2
    Cisplatin
    Imatinib
    Protein
    Cascade
    Gefitinib
    EGFR
    P
    ?
    Nucleus
    ?
  • P
    VEGF-A
    EGF
    MET
    P
    ERBB2
    P
    PI3K
    Cisplatin
    Imatinib
    Gefitinib
    Protein
    Cascade
    Protein
    Cascade
    EGFR
    P
    AKT
    PDGFR
    P
    Nucleus
  • Implications of Chromosomal Loci in the Development of Lung Cancer
    http://academic.evergreen.edu
    http://rosenblumtv.files.wordpress.com
    http://www.pharmgkb.org
  • Chromosomes
    Chromatid
    Compact structures of DNA
    Contains thousands of genes
    Centromere
    Genes
    http://staff.jccc.net/PDECELL/celldivision/chromosome1.gif
  • Single Nucleotide Polymorphisms (SNPs)
    Single variation in nucleotide base pairs
    Alleles are a result of SNPs within gene sequence
    SNP’s are the basis for lung cancer susceptibility
    http://www.dnalandmarks.com/english/pictures/what_are_snps.jpg
  • Genome-wide Association Studies
    Variation across genomes
    Genotype cases along with controls
    Find roles that genes play in disease
    Large sample sizes
    http://www.blog.speculist.com/archives/dna2two
  • Identifying Regions On Chromosomes
    6 Regions Away
    p-arm
    Centromere
    Region 5q6
    q-arm
    5
    http://images.google.com/
  • Three Regions of Association
    http://upload.wikimedia.org/
    http://upload.wikimedia.org/
  • Nicotinic Acetylcholine Receptor Genes Found on 15q24 and 15q25
    http://www.jyi.org/articleimages/88/originals/img0.jpg
    Nicotine binds and stimulates acetylcholine ion channel-linked in neurons
  • How were these regions discovered?
    (Hung et al., 2008)
  • 15q24 highly linked with smoking behavior
    (Thorgeirson et al., 2008)
  • TERT gene found on 5p15.33
    Cancerous Growth
    http://images.google.com/i
  • 5p15.33 shows similar results
    (McKay et al., 2008)
  • P
    VEGF-A
    EGF
    MET
    P
    ERBB2
    P
    PI3K
    Cisplatin
    Imatinib
    Gefitinib
    Protein
    Cascade
    Protein
    Cascade
    EGFR
    P
    AKT
    PDGFR
    P
    Nucleus
  • How Do Cells Proliferate?
  • EGFR/ERBB2 Forms a Heterodimer
    EGFR
    EGFR
    EGF
    EGFR
    ERBB2
    ERBB2
    P
    P
    Proliferation
    Protein
    Cascade
    Survival
    Nucleus
    Apoptosis
  • How Can This Lead to Cancer?
  • Mutated EGFR Leads to Cancer
    ERBB2
    EGFR
    P
    Protein
    Cascade
    Proliferation
    Nucleus
  • Mutated EGFRs have Distinct Patterns of Tyr Phosphorylation
    (Chen et al., 2006)
  • Tyrosine Kinase Inhibitors (TKIs)Gefitinib
    EGFR
    Tyrosine
    Kinase
    domain
    ATP
    TKI
    ATP binding cleft on the kinase
  • Gefitinib Suppresses Phosphorylation of Most EGFR Mutants
    (Chen et al., 2006)
  • What About the Proteins in the Signaling Pathway?
  • Effectiveness of Gefitinib is Dependent on Concentration
    (Pedersen et al., 2005)
  • High Doses of Gefitinib Decreases Proliferation of Mutated EGFR Cells
    (Pedersen et al., 2005)
  • Gefitinib has Variable Growth-Inhibitory Effects
    (Chen et al., 2006)
  • P
    VEGF-A
    EGF
    MET
    P
    ERBB2
    P
    PI3K
    Cisplatin
    Imatinib
    Gefitinib
    Protein
    Cascade
    Protein
    Cascade
    EGFR
    P
    AKT
    PDGFR
    P
    Nucleus
  • What Are the Roles of Proteins Associated with Lung Cancer?
    http://www.taconichills.k12.ny.us/webquests/noncomdisease/lungcancerpic.jpg
  • Recap
    Alanine
    N-terminus
    Tyrosine
    P
    P
    C-terminus
    P
    Nucleus
    Threonine
    Leucine
    Serine
    Lysine
  • How Does The Pathway Work?
  • Domain Attraction
    ERBB2
    P
    P
    PI3K
    Protein Cascade
    SH2
    Change in Gene Expression
    Nucleus
  • Why are EGFR and ERBB2 Special?
  • Main Contributors to NSCLC
    (Rikova et al. 2007)
  • Link between Concentration and Interaction
    (Jones et al. 2006)
    Green andBlue circles =
    Proteins involved in cascade
    Interactions
    Red lines =
    Red circles =
    EGFR Receptor
  • Other ERBB2 Associations
    No. protein receptors
    No. protein receptors
    No. protein receptors
    Affinity Threshold (nM) Affinity Threshold (nM) Affinity Threshold (nM)
    (Jones et al. 2006)
  • Are there other types of Proteins?
    http://en.wikipedia.org/wiki/Proteins
  • Other Contributors to NSCLC
    (Rikova et al. 2007)
  • Specifically, some proteins more than others
    Amount of overphosphorylated protein in samples
    Chart configured from Table1 of Rikova et al. 2007
  • Previous Research, Amplification of MET
    (Engelman et al., 2007)
  • P
    VEGF-A
    EGF
    MET
    P
    ERBB2
    P
    PI3K
    Cisplatin
    Imatinib
    Gefitinib
    Protein
    Cascade
    Protein
    Cascade
    EGFR
    P
    AKT
    PDGFR
    P
    Nucleus
  • What is the Role of Angiogenesis in Lung Cancer?
    http://www.taconichills.k12.ny.us/webquests/noncomdisease/lungcancerpic.jpg
  • Angiogenesis and VEGF
    http://www.researchvegf.com/researchvegf/multimedia/index.m#type-slides
  • Tumor Angiogenesis Is Unregulated
    VEGF-A
    PDGF-β
    VEGFR
    PDGFR-β
    Tumors
    Angiogenesis
  • P
    P
    PIP3
    P
    P85-α
    PIP2
    P110-α
    SH2
    P
    P
    P
    PI3K
    PI3K / Akt Pathway in Lung Cancer
    AKT
    P
    P
    PTEN
    Endothelial Cell
  • Akt is a Downstream Regulator
    Growth,
    Translation
    P
    mTOR
    P
    Apoptosis
    AKT
    BAD
    P
    P
    MDM2
    p53
    Nucleus
  • FLJ10540 Is An Oncogene
    Oncogene
    Proto-Oncogene
    Cancer Cell
  • VEGF-A Upregulates FLJ10540
    (Chen et al., 2009)
  • FLJ10540 Mediates Cell Migration and Invasion Through PI3K/Akt
    Giemsa-Stained Cells
    (Chen et al., 2009)
  • VEGF-A
    P
    P
    PI3K
    PIP3
    P
    P85-α
    PIP2
    P110-α
    SH2
    P
    P
    P
    FLJ10540
    Recap: FLJ10540 Missing Link in PI3K/Akt
    AKT
    P
    VEGFR
    P
    PTEN
    Endothelial Cell
  • Imatinib is a TKI
    Binds to ATP binding site of many enzymes:
    p-PDGFR-β Inhibitor
    Reduced IFP, Hypoxia
    Decreased VEGF Expression
    http://www.medicinescomplete.com/mc/martindale/2007/images/MRT9229C001.gif
  • Imatinib Targets IFP and Hypoxia
    Normal
    Tumor
    http://www.medscape.com/pi/editorial/clinupdates/2000/583/art-tu02.fig04.jpg
    http://www.nature.com/nm/journal/v7/n9/images/nm0901-987-F1.gif
  • Imatinib Reduces p-PDGFR-β and VEGF Expression
    (Vlahovic et al. 2007)
  • Imatinib Reduces Hypoxia and MVD
    Vlahovic et al. 2007
  • Imatinib Treatment
    Imatinib
    http://onctalk.com/wp-content/uploads/2008/01/angiogenesis-summary.jpg
  • P
    VEGF-A
    EGF
    MET
    P
    ERBB2
    P
    PI3K
    Cisplatin
    Imatinib
    Gefitinib
    Protein
    Cascade
    Protein
    Cascade
    EGFR
    P
    AKT
    PDGFR
    P
    Nucleus
  • Treatments
    Dr. Ross
    • Gefinitib
    Dr. Vildaver
    • Imatinib
  • Discovery of Cisplatin
    (Rosenberg et al., 1969)
  • Old Model of Cisplatin
    Nucleus
    Cisplatin
    Simple Diffusion
    P73
    P53
    NH3
    NH3
    Pt
    C-Abl
    HMG1
    ?
    G
    G
    ?
    HMG1
    Cell Cycle Arrest
    &
    Apoptosis
  • How Pt Drugs Enter Cells
    Pt
    Transporter
    protein
    Transporter
    protein
    Pt
    Pt
    Pt
    Pt
    Pt
    nucleus
    (Hall et al., 2007)
  • Crosslinks
    NH3
    nucleus
    Pt
    NH3
    G
    G
    1,2-intrastrand d(GpG)adduct
    crosslink
    Cell Cycle Arrest
    &
    Apoptosis
    (Ohndorf et al., 1999)
  • Pt-DNA adduct
    http://www.unc.edu/~shantanu/images/bkstcg_aacr07.png
  • High Mobility Group Proteins
    HMGB
    Apoptosis
    Pt-DNA Adduct
    P73
    P53
    HMGB
    (Stross et al., 2002)
  • *Nucleotides crosslinked
    Bold- nucleotides contacted by the protein
    (Ohndorf et al., 1999)
  • Phenylalanine Mutations
    (Ohndorf et al., 1999)
  • Old Model of Cisplatin
    ?
    Nucleus
    Cisplatin
    Simple Diffusion
    P73
    P53
    NH3
    NH3
    Pt
    C-Abl
    HMG1
    ?
    G
    G
    ?
    HMG1
    Cell Cycle Arrest
    &
    Apoptosis
  • 2 Distinct Regulating Mechanisms
    tyrosine kinase
    cisplatin
    mismatch repair protein
    homologue of p53
    Tumor suppresor
    (Gong et al., 1999)
  • Role of p73 in Cell Death
    MLH1-/- & Abl= less sensitive to CDDP , survived better than wild type in lower concentrations of CDDP
    P53 knockout= less sensitive to CDDP, survived better than wild type in lower concentrations of CDDP
    (Gong et al., 1999)
  • New Model of Cisplatin
    Nucleus
    Cisplatin
    Simple Diffusion
    P73
    P53
    NH3
    NH3
    Transporter
    Proteins
    P
    Pt
    C-Abl
    Cell Cycle Arrest
    &
    Apoptosis
    HMG1
    G
    G
    ATM
    HMG1
  • EGF
    ERBB2
    Cisplatin
    Imatinib
    Protein
    Cascade
    Gefitinib
    EGFR
    P
    ?
    Nucleus
    ?
  • P
    VEGF-A
    EGF
    MET
    P
    ERBB2
    P
    PI3K
    Cisplatin
    Imatinib
    Gefitinib
    Protein
    Cascade
    Protein
    Cascade
    EGFR
    P
    AKT
    PDGFR
    P
    Nucleus
  • Cocktail of Drugs
    ?
    http://www.specialityformulations.com/pcat-gifs/products-small/imatinib.jpg
    http://immunodefence.com/ii/gefitinib.gif
    http://www.themesotheliomalibrary.com/cisplatin.jpg
  • Current & Future Hypotheses
    New plasma membrane proteins have unique mechanisms and effects on NSCLC
    Gefitinib effectively inhibits particular EGFR mutants
    SiRNA could be used as a possible treatment for various mutated proteins
  • Future Studies
    How does cisplatin enter the nucleus?
    Roles and mechanisms behind the various mutated proteins involved in lung cancer
    New drugs , secondary mutations
  • Acknowledgements
    Professor Sleiter
    Schmooing yeast!!!
    ?
  • Acknowledgements
    Dr. DebBurman
  • Acknowledgements
    Mike Fiske
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