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Molecular Signaling and Cancer: Relevance to RT Bill McBride Dept. Radiation Oncology David Geffen School Medicine UCLA, Los Angeles, Ca. wmcbride@mednet.ucla.edu. Objectives:

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  1. Molecular Signaling and Cancer: Relevance to RTBill McBrideDept. Radiation OncologyDavid Geffen School MedicineUCLA, Los Angeles, Ca.wmcbride@mednet.ucla.edu

  2. Objectives: • Know how ligands work through receptors to activate phosphorylation /dephosphorylation reactions leading to gene transcription • Know how dysregulation of these pathways leads to cancer • Know how radiation-induced signal transduction pathways intersect with those altered in cancer to affect intrinsic radiosensitivity

  3. Signaling • Signal transduction evolved to allow single cells to respond to their extracellular environment. • It became more sophisticated as metazoans needed mechanisms to allow • communication between cells within tissues and between tissues to allow • morphogenesis • wound healing, • recognition and elimination of microbes, • maintenance of homeostasis.

  4. Types of Signals • External microenvironmental physiological signals • Adjacent cells, extracellular matrix, cytokines and growth factors, hormones, glucose, amino acids, ions, etc. • External microenvironmental pathological signals • Danger-associated molecular patterns (DAMPs) • Pathogen-associated molecular patterns (PAMPs) • Inflammatory and immune cells • Internal homeostatic signals • Response to DNA and mitochondrial damage, ROS, hypoxia, metabolism, etc. • Most signals are sent through ligand binding to specific cell-surface receptors, allowing multiple extracellular stimuli to be distinguished

  5. Peptide hormones Odorants Chemoattractants Neurotransmitters Taste Ligands G-proteinR Hormones Growth Factors Lipid kinases Steroid R Extracellular matrix Protein kinase R Cadherins GTP GDP Integrins Tumor necrosis Factor family TNFR family Pathogen Associated Molecular Patterns (PAMPs) Toll-likeR Nucleus Nucleus CytokineR Cytokines Damage Associated Molecular Patterns (DAMPs) GlucoseR/ Ion channels Multiple Signaling Pathways are Integrated to Make a Response • Multiple signals are integrated to generate an appropriate biological response, whether it be cell death/ survival, cell cycle arrest/ progression, glycolysis/aerobic metabolism, DNA repair/stability • Signaling pathways affect radiation responses • Radiation IS a signal

  6. Cytokine Receptor RTK (EGFR, PDGFR) P P P P P P P P P P P P The Initial Step is Receptor Activation by Ligand-Induced Oligomerization Inactive Receptors Activated Receptors ATP ADP autophosphorylation • leads to activation of receptor kinases or conformational changes that allow adapter proteins that bind to activate cascades • Receptors can co-associate with others to synergize eg ErbB1 and 3 - may be important in cancer escape from targeting

  7. Signals Change mRNA and Protein Levels • Transcriptional activation • Post-transcriptional mRNA stabilization • AU rich UTRs • Translational control mechanisms • Post-translational protein destabilization and stabilization • phosphorylation, ubiquitination, acetylation, oxidation, nitrosylation • Protein degradation • Stabilization of mRNA and protein expression allow rapid responses - immediate early genes - fos, jun, GM-CSF, TNF-, p53, IkB, etc. IR can cause ALL of these!

  8. Major Players - Kinases • Tyrosine kinases (100 genes) • Growth Factor Receptors (RTKs; 60 genes) • Cytoplasmic (35-40 genes) Jak, Src, Fak, Tec… • Serine/threonine kinases (400 genes) • MAP Kinases, TGF-R, PKC, ATM • Dual specificity kinases • MEK Phosphatases (eg PTEN, CDC25) control phosphorylation.

  9. Epidermal Growth Factor Receptor family erbB1 (c-erbB) erbB2 (neu) erbB3 erbB4 Fibroblast Growth Factor Receptor family FGFR-1(fig) FGFR-2(K-sam) Platelet Derived Growth Factor Receptor family CSF-1R (c-fms) SLF R (c-kit) Insulin Growth Factor Receptor Family IGFR-1 (c-ros) Neurotrophins NGFR (trk-A) BDNFR (trk-B) NT3 R (trk-C) A Few Examples - RTKs

  10. Phosphorylation • Alters activity of enzymes initiating cascades • eg MAP kinase pathway initiated by activation of EGFR by auto-phosphorylation. • Induces DNA binding • STAT and c-jun transcriptional activities • Changes subcellular localization of proteins • e.g. recruitment of adaptor to activated receptors, nuclear localization of hormone receptors • Changes protein stability • phosphorylation leads to degradation or stabilization - p27, IkB, p53, etc .

  11. Molecular Features of Cancer Mutations in molecular signaling pathways leading to • Self-sufficiency in growth factor signaling (ligands or receptors) • Loss of response to anti-proliferative signals • Evasion of programmed cell death • Increase in replicative potential (telomeres) • Promotion of tissue invasion and metastasis • Sustained angiogenesis • Amplified by DNA repair abnormalities and genomic instability Hanahan D, Weinberg RA, Cell 57-70, 2000. Overall decrease in cell loss factor

  12. “Driver” mutations in protooncogenes give oncogenes that generally cause gain in function. • Tumor suppressor genes are the “brakes”. Mutations in these cause loss of function and generally both alleles need to be affected. • Activated oncogenes and loss of tumor suppressor genes cause replication stress and increased DNA damage, which results in tumor progression

  13. Tumor cells become “addicted” to the mutated molecules and pathways they need for their existence • This is good news because targeting these critical molecules can have dramatic consequences • The bad news is that the mutation rates often allow variants to escape • Although the steady state of the tissue cells is disturbed, there is still a lot of cell loss. • Cancer stem cells exist that may be a small minority of the population. They may not be the targeted by the chosen therapy. Rapid tumor regression may not mean much if it represents loss of the non-stem cell population • Cancer stem cells are responsible for tumor regrowth and treatment failure

  14. Oncogenes • The first oncogene (src) was discovered in 1970 in a chicken retrovirus. In 1976, Bishop and Varmus demonstrated that oncogenes were defective proto-oncogenes that coded for normal growth and differentiation proteins (‘the enemy within”), for which they received the Nobel Prize in 1989. • Oncogenes are “driver” mutations that encode • Receptor/cytoplasmic tyrosine kinases (EGFR, PDGFR, Ras/MAPK) • Ser/thr kinases (AKT, mTOR) • Lipid kinases (PI-3K) • Transcription factors (c-MYC, STATs, c-JUN, c-FOS) • Cyclins/CDKs (Cyclin D) • Regulators of protein stability (MDM2) • Anti-apoptotic factors (BCL-2, BCL-XL) • They gain function by • Domain deletion (EGFRvIII, Her2) • Point mutation (Ras) • Translocation (BCR-Abl, Myc) • Altered subcellular localization (BCR-Abl) • Gene amplification (Myc, EGFR, Her2)

  15. Oncogenic Mutations in Cancer H-Ras K-Ras N-Ras Neu EGFR Increased expression int-1 int-2 mos Altered protein Myc K-Ras Myb RelA EGFR Point mutation Amplification Insertion Protooncogene Translocation Deletion Normal protein Altered protein EGFR, (ERB-B1), NF-B Altered Protein Abl, Trk Increased expression C-Myc, Bcl-2

  16. OLI OLI S/TK SH3 SH2 TK DB AB Philadelphia Chromosome Bcr (160KDa) (Breakpoint cluster region) OLI OLI S/TK DH Rac GAP NTS Abl (140KDa) SH3 SH2 TK DB AB Bcr-Abl ALL (190KDa) CML (210KDa/230 KDa) JAK1/2 Crk-L Grb2 Nowell and Hungerford (1960) t(9;22)(q34;q11) CML - 95% ALL, 25-30% in adult and 2-10% in pediatrics Abnormal signaling and localization PI-3 kinase STAT3 STAT5 Sos Akt Ras Cyclin D1,D2,D3 Bcl-xL ERK1/2

  17. Imatinib/Gleevec/STI571 • Druker, Sawyers and Talpaz showed that Gleevec inhibits proliferation of CML • Inhibits Abl by binding to the ATP-binding site in the kinase domain • Relapse as a result of the outgrowth of leukemic subclones with resistant BCR-ABL mutations - treated with dasatinib

  18. 1 143 434 407 413 355 366 MB I MB II Myc LZ HLH Transactivation Domains DNA Binding and oligomerization 1 151 MB= Myc Boxes HLH= Helix-Loop-Helix Max LZ HLH BR= Basic Region LZ= Leucine Zipper Myc-induced Oncogenesis • Mechanisms of oncogenic activation • 70% of cancers have deregulated Myc • Chromosomal translocations increase c-Myc transcription (Burkitt’s lymphoma and other lymphoid malignancies) • Gene amplification (AML, lung, breast, colon, brain, prostate) • Point mutations increase transactivation function (breast, ovarian, colon)

  19. C-Myc gene k,l,m loci P1 P2 t(8;14) J Ei Cm Ca1 P1 P2 t(2;8) MAR Ei Ck Translocation P1 P2 t(8;22) Cl El C-myc Translocations in Cancer • Translocations link the c-Myc gene to a region of transcriptionally active DNA • This increases c-Myc expression levels and induces aberrant proliferation • In contrast to BCR-Abl, c-Myc translocations DO NOT alter the protein structure; they increase expression levels of the WT gene and protein

  20. Translocation Genes Disease t(8;14)(q24;q32) c-myc/IgH Burkitt’s lymphoma Igk/c-myc Burkitt’s lymphoma t(2;8)(p12;q24) Burkitt’s lymphoma c-myc/Igl t(8;22)(q24;q11) Diffuse large cell lymphoma c-myc/IgH t(8;14)(q24;q32) c-myc/TCRa,b T-cell acute lymphoblastic leukemia t(8;14)(q24;q11) t(8;14)(q24;q32) c-myc/IgH Multiple myeloma Igk/c-myc t(2;8)(p12;q24) Multiple myeloma t(8;22)(q24;q11) c-myc/Igl Multiple myeloma C-Myc translocations and disease

  21. Tumor Suppressor Genes • Tumor suppressor genes are the ‘brakes’ that protect cells from carcinogenesis. A.G. Knudson first proposed for Retinoblastoma (Rb) that loss of both alleles is required for loss of function. This is true for most but not all Ts genes. • Hereditary • Peaks at 6 months of age • Both eyes • Heterozygous +/- • Second cancers 36% cumulative risk at 50 yrs of age • Non-hereditary • Peaks at 2 years of age • One eye affected • +/+ • Second cancers 6% cumulative risk at 50 yrs of age

  22. Loss of function mutations include genes encoding • Phosphatases (eg. PTEN) • Transcription factors/repressors (p53) • Repair genes (BRCA1/2, MSH) • Cell cycle inhibitors (Rb) • Regulators of protein stability (c-Cbl) • Apoptosis inducers (Bax, Bad) • Leading to • Lack of cell cycle arrest • Decreased apoptosis • Increased metastasis

  23. Multiple Mutations are Required for Oncogenesis • Transfer of a single oncogene to a normal cell is normally not sufficient to transform it • Loss of one allele of a Ts gene is insufficient • Cancer frequency increases with age, suggesting that transformation of cells requires the accumulation of multiple mutations • Most oncogenes can induce both growth and apoptosis, indicating that transformation requires one mutation that enhances cell growth and another that inhibits cell death (oncogene “cooperation”). Examples of “two hit” gene pairs in tumors: Ras/p16 BRCA1/p53 p27/Rb Myc/p53 Myc/Ras

  24. Oncogene Cooperation (validation of the “two hit” hypothesis) Expression of c-myc or ras alone fails to transform cells C-myc Ras P16 P19 Arf p53 Apoptosis Senescence Transformed focus Expression of both c-myc and ras is transforming

  25. 10 0 2 4 6 8 Rat -1/ bcr-abl Rat -1/v- fes Rat -1/c-myc Rat -1/v-mos Rat -1/wt-ras Oncogene Expression and Radiation Resistancy Dose (Gy) 1 S.F. 0.1 0.01 Rat -1/v-Ha-ras Rat -1 Chiang, CS Molecular Diagnosis 3; 21, 1998 Oncogene-induced radioresistancy does not need transformation but is based on the signal transduction pathways that are activated, and interaction between oncogenes may negate each other

  26. A Multi-step Process in Colorectal Cancer Normal Epithelium APC(adenomatous polyposis coli)-catenin Small Adenoma Increasing Genetic Instability K-Ras/BRAF LargeAdenoma SMAD4/TGF-RII PI3K3CA/PTEN TP53/BAX Carcinoma ? Metastasis

  27. Percentage of Human Tumors Overexpressing EGFR Percentage of tumors Tumor type Bladder Breast Cervix/uterus Colon Esophageal Gastric Glioma Head and neck Ovarian Pancreatic Renal cell Non-small-cell-lung 31-48 14-91 90 25-77 43-89 4-33 40-63 80-100 35-70 30-89 50-90 40-80

  28. Glioblastoma multiforme Normal loss of 17p, TP53; PDGF-aR overexpression Grade II Loss of RB, 18q, 9p/IFN/p16; CDK4, MDM2 amplification Grade III EGFR amplification/mutation PDGF-a/b overexpression, loss of PTEN phosphatase on chr. 10 Grade IV GBM • About 40% of glioblastomas show amplification of the EGFR gene locus and about half of these express a mutant receptor (EGFRvIII) that is constitutively active due to an in-frame truncation within the extracellular ligand-binding domain. • EGFRvIII confers radioresistancy • 15-20% of glioblastoma patients respond to small-molecule EGFR kinase inhibitors, but only if they have an intact PTEN (phosphatase and tensin homolog). • Inhibition of mTOR, which is downstream from PTEN, with rapamycin helps.

  29. P PTEN P P P P P P P binds phosphotyrosine residues SH2 SH3 binds proline-rich sequences binds lipid ligands (products of PI-3K) PH Glucose Amino acids EGFR GLUT1 PIP2 PIP3 P P sos Glucose Glucose-6-P Glycolysis SH3 PH Mutant Ras Akt PKA PI-3K Grb2 P SH2 P GDP sos SH2 x PIP2 PIP3 LKB1 Ras GTP P110 Raf-1 Src AMPK MEK ERK1 ERK2 MAPK/ERK signaling rapamycin BAD NF-kB FKHD GSK3 MDM2 mTOR p27 FasL p53 cell death/survival cell cycle arrest/progression DNA repair/misrepair cell metabolism

  30. Q61L P Src 32 40 192 1 MEK GTPase GTP binding ED GTP binding HVR ERK1 ERK2 ERK1 ERK2 MEK CAAX Box (prenylation) G12V Ras Oncogenic Mutations EGFR P sos Tethers Ras to membrane Grb2 P GDP Farnesyl Transferase Inhibitors sos x Ras GTP Raf-1 • One of the most commonly mutated genes • G12V and Q61L are both involved in GTP binding • Both mutations stabilize the GTP-bound form of Ras • Both result in constitutive MAPK signaling

  31. Ras Mutations in Human Tumors *K=Kirsten; H=Harvey; N=neuroblastoma Mutation frequency % Predominant Ras isoform* Cancer or site of tumor Non-small-cell lung cancer (adenocarcinoma) 33 K K Colorectal 44 K Pancreas 90 Thyroid H,K,N 53 Follicular H,K,N 60 Undifferentiated papillary 0 Papillary K,N 43 Seminoma N 13 Melanoma H 10 Bladder 30 N Liver H 10 Kidney N,K 40 Myelodyplastic syndrome N Acute myelogenous leukemia 30

  32. Genetic Mutations Glioblastoma 25-75% Gastric 28% Melanoma 20-30% NHL 10% Breast 15% Prostate 30% Endometrium 40-80% Ovary 5% Lung 22% Bladder 10% Gene Methylation Glioblastoma 35% Colorectal 8% Invasive Breast 48% Melanoma 62% Thyroid 50% Endometrium 20% Prostate 50% The PTEN Ts Gene • PTEN Mutations are linked to • Cancer (eg Cowden’s syndrome), invasiveness, metastasis • resistance to Herceptin, Vincristine, Adriamycin, 5-fluourouracil, Cisplatin

  33. Retinoblastoma Gene Mutations in Cancer Retinoblastoma 70% Small Cell Lung Carcinoma 80% Non-Small Cell Lung Carcinoma 20-30% Osteosarcoma >50% Multiple Myeloma 30% Mitogens Sherr (2000) Cancer Research 60:3689-3695 Cyclin D CDK 4/6 E2F P E2F Rb P + Rb P CDK 2 Cyclin E S phase entry E2F Cyclin E Cyclin E gene

  34. TP53 (p53) • Transcription factor that also binds DNA DSBs • Degraded by binding mdm2 • Mutated in >50% human cancers, in DNA binding domain • Activated by IR through ATM, DNA-PK, etc. • Increases p21 (cell cycle arrest) and Bax (apoptosis) expression • TP53 -/- mice are sensitive to DNA damage and have high incidence of tumors • TP53 mutated tumors are generally more aggressive cancers and more radioresistant P53 protein 102 363 1 50 292 323 356 393 TAD MDM2 DNA binding TET CTR Ser33 Ser376 Ser15 Ser37 Ser392 ATM ATR Ser20 DNA-PK ATR ATM ATR Chk1 Chk2 Phosphorylation sites Decreased MDM2 binding Increased transcriptional activation MDM2 237 260 289 333 108 1 489 nls p53 binding Ser395 II III IV I ATM (inhibition of p53 nuclear export)

  35. 1.4 1.2 1.00 SKOV 1.0 S.F. 0.10 0.8 SKOV/P53 0.6 0.01 4 2 0 0.4 DOSE (Gy) 0.2 0.0 0 10 20 30 40 50 TP53 Gene Transfer Radiosensitizes Tumor AdVluc+Irrad. AdVp53 control Tumor Diameter (cm) AdVp53 +irr. irrad. irrad. xxx xxx Time (days) In Vitro In Vivo

  36. What are the Rules? • Cancer is associated with deregulation of the same signaling pathways as determine intrinsic cellular radiosensitivity • Activation of cell survival/cell cycle progression pathways generally result in increased radioresistance • Activation of pro-apoptotic/cell cycle arrest pathways generally radiosensitize • The deregulated signaling pathways to which the cancer becomes “addicted” will provide the best targets for modifying radioresistance

  37. Intrinsic radioresistancy is driven in part by genetically determined signaling pathways Cancer-associated mutations will affect responses to radiation Oncogenic stress may activate DNA damage responses There is a link between DNA repair defects and cancer Molecular staging of cancer may predict response Summary

  38. Microarrays Gene Microarray Tissue Microarray Normal Tumor 40,000 probes for 20,000 genes Compare with common reference sample Cy3 Cy5 labeled nucleotides For staging, the aim is to define a Prognosis Classifier of <100 genes

  39. Improved Molecular Staging • Current clinical and pathologic criteria are inadequate - there is marked variation in response to therapy amongst apparently homogeneous cancers • The hope is that molecular classification will provide more accurate criteria for staging cancer and that this will be more predictive of response to therapy

  40. Gene Microarray Analysis • Patient samples are sorted on the basis of similarity in expression across a set of specified genes using hierarchical clustering algorithms • For example • Red/black/green may represent above/average/below average expression • Dendrograms are formed to express relatedness • short branches more related than long

  41. Lung Carcinoma 67 tumors, 56 patients Garber et al. PNAS 98 13784 2001

  42. 612 608 CDK binding site S B A N E2F C LXCXE 567 5 230 252 356 373 807 249 795 S/T phosphorylation sites 788 826 780 821 811 Retinoblastoma Protein (pocket proteins) Target for viral oncoproteins: Adenovirus E1A SV40 Large T Human Papilloma Virus E7 Viral gene products as well as spontaneous and germline mutations disrupt the Rb-E2F interaction, resulting in increased cell cycle progression and transformation.

  43. HPV • HPV is the most common sexually transmitted disease • HPV infection is an essential factor in cervical carcinoma and is associated with esophageal, oropharyngeal, and anal cancer as well as penile, vulvar and vaginal cancer. • HPV-16 is the most common HPV type associated with a malignant phenotype regardless of origin. • What is the role of vaccines - Cervarix” and “Gardasil”?

  44. Invasive cancers show Increased aerobic glycolysis (Warburg effect), even in vitro Increased glycolysis through hypoxia Up-regulated glucose transporters (esp. GLUT1 and3) and hexokinases I and II Increased uptake of FdG Acidification of extracellular space through H+ production as a metabolic product of glycolysis Biochemical Features of Cancer Warburg hypothesis 1924 “the prime cause of cancer is the replacement of the respiration of oxygen in normal body cells by a fermentation of sugar." Otto Warburg: The Nobel Prize in Physiology or Medicine 1931

  45. Glucose metabolism in mammalian cells. Afferent blood delivers glucose and oxygen (on haemoglobin) to tissues, where it reaches cells by diffusion. Glucose is taken up by specific transporters, where it is converted first to glucose-6-phosphate by hexokinase and then to pyruvate, generating 2 ATP per glucose. In the presence of oxygen, pyruvate is oxidized to HCO3, generating 36 additional ATP per glucose. In the absence of oxygen, pyruvate is reduced to lactate, which is exported from the cell. Note that both processes produce hydrogen ions (H+), which cause acidification of the extracellular space. HbO2, oxygenated haemoglobin. Gatenby and Gillies, Nature Rev Cancer Cancer cells prefer aerobic glycolysis, even though it is less efficient, because it is faster at generating ATP, which explains the Warburg effect. One result is up-regulation of glucose transport, which is why FDG-PET works. PI3-K, AKT, mTOR, and AMPK are major players in the metabolic pathway driving glycolysis.

  46. Leucine Glutamine High ATP High AMP hypoxia Low glucose Exercise, TNF 2-deoxygluc AICAR metformin glucose PI3K PIP3 NO PIP AKT PTEN STAT JAK hexokinase Pim1/2 Ribose+NADPH oxidase G-6P TSC2 AMPK BAD NAD++ADP RHEB RHEB Apoptosis GDP GTP MTOR NADH+ATP +Pyruvate LDH-A lactate +NAD+ 4EBP1 Cap-dep translation EF2 EIF4E Autophagy Acetyl CoA Acetyl CoA proteins lipids citrate P70 S6K Ribosome function TCA mitochondria Fatty acids Amino acids ADP ATP

  47. Hypoxia/reperfusion selects for epigenetic and genetic changes that promote • Glycolysis • Glucose uptake • Intracellular pH homeostasis (H+-ATPases) • Cell survival e.g. mtp53, NF-B, HIF-1

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