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Lung cancer 2011 talk-for 17-10-2011-17-10-2011

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MAGE genes and MAGE antigens

MAGE genes and MAGE antigens

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  • 1. From metagenes to biomarkers, a possibleapproach for prognosis and personalized medication through GWAS of lung cancer (NSCLC) Cheng Luo, Ph. D University of Tartu
  • 2. 1, Metagene MAGEs obtained from GWAS (or geneexpression profiling of whole genome) of lungcancer are immunotherapy target.2, The prognosis influenced by EGFR and VEGFmutation and variation.3, COX-2/Keap1/Nrf2/ARE(s) antioxidant pathwaysin lung cancer (NSCLC).4, Acetaldehyde test in A549, an antioxidant assayfrom in vivo to in vitro.
  • 3. 1, Metagenes obtained from GWASare possible to be genetic /diagnosis /prognosis biomarkers, and immunotherapy target
  • 4. Metagenes associated to survivals Genes up-regulated in NSCLC Gene ID adj. p-value Fold change SPP1 2,76E-17 14,52 MMP1 5,20E-11 10,74 KRT6A 3,85E-05 10,39 KRT17 1,86E-06 9,44 CTHRC1 2,76E-17 7,62 S100A2 2,21E-05 7,44 MMP11 1,29E-11 7,14 TMPRSS4 1,04E-08 6,85 CDC20 1,59E-13 6,82 KRT6B 7,41E-04 6,15 Genes down-regulated in NSCLC Gene ID adj. p-value Fold change SFTPC 7,13E-10 24,43 FCN3 6,77E-17 16,71 FABP4 3,28E-14 13,54 C19ORF59 5,43E-20 12,51 TMEM100 1,65E-21 12,11 CYP4B 1 1,35E-12 11,41 CLIC5 9,37E-21 10,99 SFTPD 4,18E-08 10,75 SFTA2_HUMAN 4,02E-07 10,59 CLEC3B 9,66E-22 10,23 Urgard E. et al. 2011
  • 5. MAGE family, PTGS2 (COX-2), Nre2l2 (Nrf2) and SOD1 (ARE genes)
  • 6. Metagene: MAGEA9B,MAGEA10 (melanoma,ageing scenery) Expression anddemethylation ofMAGE genes occuronly in cancer cells(except testis)MAGE expression isknown to be activatedby promoterdemethylation.
  • 7. Dendrogram Some of mouse andrepresentation of the human MAGE gene smultiple alignment are identicalbetween MAGEconserved domains ofhuman and mouse MAGEproteins. Kufer P. et al., 2002, Heterogeneous Expression of MAGE-AGenes in Occult Disseminated Tumor Cells A Novel Multimarker Reverse Transcription- Polymerase Chain Reaction for Diagnosis of Micrometastatic Disease Chromez P. et al 2001, An Overview of the MAGE Gene Family with the Identification of All Human Members of the Family
  • 8. MAGE genes: transcriptional factorsBarker PA et al: The MAGE proteins: emerging roles in cell cycle progression, apoptosis, andneurogenetic disease. J Neurosci Res 67:705-712, 2002.Ohman FK et al: The melanoma antigen genes – any clues to their functions in normaltissues? Exp Cell Res 265(2): 185-194, 2001.Chromez, et al 2001, An Overview of the MAGE Gene Family with the Identification of AllHuman Members of the Family , Cancer Research
  • 9. Frequent MAGE Mutations in Human Melanoma: Distribution of mutations in the MAGE genesCaballero OL et al. Frequent MAGE Mutations in Human Melanoma, 2010 PLOS One
  • 10. Swiss CohortCaballero OL et al. Frequent MAGE Mutations in Human Melanoma, 2010 PLOS One
  • 11. Australiacohort
  • 12. Globalcohorts byliteratures
  • 13. Chicago cohortKufer P et al. 2002, Cancer Research
  • 14. Genetic make-up of MAGE genes of NSCLC of Estonia cohorts?
  • 15. MAGE Expressions Mediated by Demethylation of MAGE Promoters Induce Progression of Non-small Cell Lung Cancer YANAGAWA N. et al. 2011Correlation between MAGE expression and overall survival of 67 NSCLC patients using the Kaplan-Meier method.The patients with any expression had poorer prognosis than those with no expression.
  • 16. MAGE (Melanomaassociated antigen)participate theapoptosis!!!Nasarre P. et al., 2010, Guidance molecules in lung cancer. Cell AdhMigr. 4(1):130-45.
  • 17. MAGE genes in clinic Jheon S. et al. Lung cancer detection by a RT-nested PCR using MAGE A1—6 common primers, Lung Cancer 2004 Conclusion MAGE A1—6 RT-PCR showed high sensitivity and specificity in various respiratory specimens, and for lung cancer detection, based on sputum samples, MAGE is probably the best of the known tumor markers. The satisfactory conclusion of workaimed at determining the causes of false positivity,should ensure that MAGE A1—6 RT-PCR becomes an effectively diagnostic tool for lung cancer. TANG Yan, XIA Da Wen, LI Ya Rong, et al. 2010, Measurement of Lung Cancer Cells in the Sputum and Peripheral Blood by the MAGE A1-6 mRNA as Specific Markers
  • 18. Selvan SR et al. 2010, Establishment of stable cell lines forpersonalized melanoma cell vaccine Table 4 Various combinations of six different TAA distributions in melanoma cell linesPresence Combination of No. of Presence of Combination of No. of TAAs expressed TAAs expressed byof no. of TAAs by melanoma cell cell lines no. of TAAs melanoma cell lines cell lines(absence of lines (absence ofspecific TAAs) specific TAAs)6 S-100/MAGE-1/Mel-5/HMB-45/ Melan-A/Tyrosinase 9 3 (S-100/MAGE-1/Tyrosinase) Mel-5/HMB-45/Melan-A 55 (S-100) MAGE-1/Mel-5/HMB-45/Melan-A/ 3 (S-100/MAGE-1/Mel-5) HMB-45/Melan-A/Tyrosinase 4 Tyrosinase 19 3 (Mel-5/HMB-45/Melan-A) S-100/MAGE-1/Tyrosinase 25 (MAGE-1) S-100/Mel-5/HMB-45/Melan-A/ 3 (S-100/Mel-5/Melan-A) MAGE-1/HMB-45/Tyrosinase 1 Tyrosinase 85 (Mel-5) S-100/HMB-45/Melan-A/MAGE- 3 (S-100/Mel-5/Tyrosinase) MAGE-1/HMB-45/Melan-A 1 1/Tyrosinase 3 3 (S-100/MAGE-1/HMB-45) Mel-5/Melan-A/Tyrosinase 15 (HMB-45) S-100/MAGE-1/Mel-5/Melan-A/ 3 (S-100/HMB-45/Tyrosinase) MAGE-1/Mel-5/Melan-A 1 Tyrosinase 1 3 (MAGE-1/Mel-5/Tyrosinase) S-100/HMB-45/Melan-A 14 (S-100/MAGE-1) Mel-5/HMB-45/Melan-A/ 2 (MAGE-1/Mel-5/HMB-45/ Melan-A)S-100/Tyrosinase 3 Tyrosinase 20 2 (S-100/Mel-5/Melan-A/Tyrosinase)MAGE-1/HMB-45 14 (S-100/Mel-5) MAGE-1/HMB-45/Melan-A/ Tyrosinase 6 2 (S-100//MAGE-1/Mel-5/HMB-45)Melan-A/Tyrosinase 14 (S-100/HMB-45) MAGE-1/Mel-5/Melan-A/Tyrosinase/ 2 2 (S-100/MAGE-1/HMB-45/Melan-A)Mel-5/Tyrosinase 14 (MAGE-1/Mel-5) S-100/HMB-45/Melan-A/Tyrosinase 1 1 (S-100/Mel-5/HMB-45/Melan-A/Tyrosinase)MAGE-1 34 (MAGE-1/HMB-45) S-100/Mel-5/Melan-A/Tyrosinase 1 1 (S-100/MAGE-1/Mel-5/HMB-45/Melan-A)Tyrosinase 24 (Mel-5/HMB-45) S-100/MAGE-1/Melan-A/Tyrosinase 1 1 (S-100/MAGE-1/HMB-45/Melan-A/Tyrosinase)Mel-5 1Total cell lines 106 0 (S-100/MAGE-1/Mel-5/ HMB-45/Melan-A/Tyrosinase)None 7TAA, tumor-associated an Total cell lines 106, TAA, tumor-associated an
  • 19. Increased expression of MAGEA9B, MAGEA10 for longger survival of NSCLC in Estonia Cohort Two situations for the expression of MAGE genes in cancers: the expression of MAGE genes may block apoptosis, but as cancer-specific antigen, the expression may stimulate immune response, particularly cancer – specific IgG that could attack cancer cells self, or by vaccination. High expression of meta gene was associated with increase in the survival time (Urgard E. et al 2011). This fits with immune theory.
  • 20. 5.3. MAGE-A3 vaccineMAGE-A3 is a tumor-associated antigen that is notexpressed in most normal cells [31,61]. Approximately 35–50% of lung cancers express MAGE-A3, and expression hasbeen associated with poor prognosis [31]. This providedthe rationale for the development of a TCV comprisingMAGE-A3 recombinant protein combined with the AS02Bimmunoadjuvant.In a randomized phase II study, 182 patients withcompletely resected, MAGE-A3-expressing, stage IB or IINSCLC were randomly assigned to receive postoperativeMAGE-A3 vaccine or placebo (2:1) [62]. The HRs fordisease-free interval, disease-free survival (DFS) and overallsurvival were 0.74 (95% CI 0.44–1.20), 0.73 (95% CI 0.45–1.16) and 0.66 (95% CI 0.36–1.20), respectively, in favor ofthe MAGE-A3-treated patient group. The ongoing phase IIIMAGRIT (MAGE-A3 as Adjuvant, Non-Small Cell LungCancer Immunotherapy) trial, initiated in 2007, plans toenroll approximately 2300 MAGE-A3-expressing patientswith resected stage IB, II, or IIIA NSCLC who will berandomized to vaccine or placebo, again in the adjuvantsetting. The administration of adjuvant platinumbasedchemotherapy for the treatment of the current NSCLC isallowed between surgery and randomization. The study ispowered for DFS as the primary endpoint in the totalpopulation and in the cohort without postoperativechemotherapy [63]. Mellstedt H. et al 2011
  • 21. Immunotherapy in NSCLC
  • 22. Lung Cancer Vaccine Shows Promise http://www.gsk.com/
  • 23. Other Metagene: NLRP2 Inflammation, proliferation,tu morigenesis. COX-2
  • 24. Metagene:CYP3A5 Detoxification, antioxidation
  • 25. Metagene: AKR1B1
  • 26. Summary and next: Likely, MEGA gene families, NLRP2, CYP3A5, AKR1B1, KEAP1 are decisive genes for survival of lung cancer for Estonia cohort. MAGE genes mutation frequency and expression distribution (PCR and Sequencing) for NSCLC in stock (Tarmo has total RNA) of Estonia cohort. MEGA(s) are cancer/testis specific antigen (recognized by HLA-A1 of CTL), high expression of MEGA genes make them ideal targets for immunotherapy for NSCLC patients: (polysaccahride, or other immunojuvants containing “syrup”) spray and vaccine!!! Next: Analyze MAGE genes’ expression (lung tissue, or sputum and peripheral blood) for further determination of MAGE gene expression, methylation/demethylation’s impacts on NSCLC for Estonia Cohorts, and for personalized medication, or vaccine preparation.
  • 27. 2, The prognosis influenced by the EGFRand VEGF mutation and other geneticmake-up, or variationToday major personal medication or translational medicineare from EGFR and VEGFEGFR and VEGF were not among 599 genes which were down-regulatedand 402 genes which were up-regulated in Urgard E. et all 2011. However, typically the genemutation, or methylation, or other tissue-derived molecular information ofEGFR and VEGF has been combined with individuals personal medical history,family history, and data from imaging, and other laboratory tests for bettereffective treatments for a wider variety of conditions.
  • 28. EGFR (Her-1), and VEGF, a clinicexample of translational medicine To determine whether a solid tumor, such as for the lung (non small cell), head and neck, colon, pancreas, or breast, the mutation positive or negative for EGFR, helps to guide treatment and determine prognosis When one have been diagnosed with certain invasive cancers, more and more doctors want to see if EGFR is being over-expressed in the tumor, in order to choose what kind of chemotherapies.
  • 29. EGF (R), VEGF and others promote angiogenesis
  • 30. Bifunctional inhibitor of VEGF and EGFR Ryan AJ. et al. 2005
  • 31. If VEGF mutation or overexpressionInvassion Alitalo K. et al 2002
  • 32. Gene mutation may increase survivalTKI:tyrosinekinaseinhibition,RR:responserate, TTP:time toprogression
  • 33. VEGF expression associated with tumor growth and survival (mutation, and silence by methylation)(A) Overall survival in patients with VEGF-negative and VEGF-positivetumours (P<0.02). Fondevila C. et al 2004, Santini D. et al 2005
  • 34. Summary:Mutation or gene silence by methylation ordemethylation may help the therapy andprognosis, so it is good to know the patients’genetics make-up in clinic.
  • 35. 3, COX-2/Keap1/Nrf2/ARE(s) antioxidant pathways in lung cancer (NSCLC)
  • 36. COX-2/Keap1-Nrf2/ARE antioxidant pathway (Global Keap1 methylation in cancer tissues) COX-2The dark side ofNrf2:1, Release of Nrf2 EFOXcause large numberof antioxidant geneexpression, whichmake cancer cellgrow faster.2, Nrf2 enhancesresistance of cancercells tochemotherapeuticdrugs, Luo C. et al Med. Hypo., 2011
  • 37. Methylation of selected genes (Method 1) Kaie Kirotar (UT) provided methylation array data matrix
  • 38. Method 2: Hypermethylation of Keap1 in41-79 year old NSCLC patients (A549 (NSCLC) cellcontains several mutations in Keap1 gene) Kaie Kirotar (UT) provided methylation array data matrix
  • 39. Cell division, proliferation, tumorigenesis EGFR Nrf2COX-2 dependentEFOX on CUL-Keap1, 2010 Antioxidation
  • 40. COX-2, from inflammation to antiinflammation COX-2 plays a crucial role in inflammation, invasion, development and metastasis of non-small cell lung cancer (NSCLC) COX-2 inhibitor was used in cancer therapy because painkilling and shrinking blood vessels, but dangerous for cardiovascular. But today COX-2 antiinflammatory roles may weigh more than inflammatory roles because of its EFOX molecules trigger ARE genes.
  • 41. Nrf2, an intrigue gene in cancer therapy Nrf2 was regarded as a protective transcriptional factor because it switch on antioxidant response elements Nrf2 maintain an internal antioxidant and antiinflammatory system. Nrf2 constitutively overexpressed in most cancer cells, which inhibit apoptosis, and promote cell proliferation. Reactive oxygen species (ROS) induce Nrf2, which may inhibit cell proliferation in normal cells, but promote cell proliferation and inhibit apoptosis in cancer cells.
  • 42. Schematic presentation of the roles that Nrf2 plays in cancer cell proliferation and cancer cell resistance to anticancer drugs. COX-2’ EFOX (Green risk!)Homma S et al. Clin Cancer Res 2009;15:3423-3432
  • 43. KEAP1 mutations - dysfunction inLung-Tumor-Derived Cell Lines and Patients
  • 44. Expression of Keap1 and Nrf2 inNSCLC cell lines Low Keap1, high Nrf2 NE: nuclear extracts, TP: total protein Singh A. et al., 2006
  • 45. The capacity of Nrf2 antioxidant ROS level become low in Keap1 -/- knockout mice because Nrf2 activated Nrf2, a green risk! Singh A . et al. 2010
  • 46. Summary: Internal antioxidants maintained by Nrf2/AREAcetylationAnddeacetylation SOD
  • 47. 4, Acetaldehyde in A549, an antioxidantassay from in vivo to in vitro
  • 48. Experimental approaches TCA cycle carcinogenEthanol is first oxidised to ethanal then ethanoic acid. Note that while different enzymes can beused in the first step, only aldehyde (ethanal) dehydrogenase (ALDH) is used in the second
  • 49. Jogurteista ja lasten hedelmäsoseista löytyi korkeita pitoisuuksia asetaldehydiä Turun Sanomat 7.2 2010 05:00:11– Tutkin nykyään laboratoriossa, mitä lastenlapseni syövät. Viili onturvallisempi kuin jogurtit, professori Mikko Salapuro sanoo.Professori Mikko Salaspuro on huolissaan suomalaistenelintarvikkeiden asetaldehydipitoisuuksista. Maailman terveysjärjestönWHO:n alainen kansainvälinen syöväntutkimuslaitos luokitteliasetaldehydin viime lokakuussa ihmisille syöpää aiheuttavaksi aineeksialkoholin juomisen yhteydessä. Se nousi ylimpään riskiluokkaan, johonkuuluvat myös tupakka ja asbesti.Suun ja suoliston bakteerit ja hiivat muuttavat alkoholin syöpääaiheuttavaksi asetaldehydiksi. Ainetta on myös alkoholijuomissa jatupakassa.Lisäksi asetaldehydiä löytyy useista tavallisista elintarvikkeista. Sitämuodostuu käymisellä valmistettuihin tuotteisiin, kutenmaitovalmisteisiin, soijaruokiin, säilöttyihin vihanneksiin, etikkaan,kotikaljaan ja simaan.Asetaldehydiä käytetään myös elintarvikkeiden aromiaineena. Ainettalisätään esimerkiksi hedelmämehuihin, virvoitusjuomiin, jälkiruokiin jamaitovalmisteisiin. Pitoisuuksia ei tarvitse ilmoittaa tuoteselosteessa.Asetaldehydiä muodostuu myös sisäsyntyisesti joihinkin hedelmiin,kuten omenoihin, päärynöihin ja marjoihin. Sitä käytetään myösmarjojen ja hedelmien säilöntäaineena.Mikko Salapuron työryhmä on tutkinut laboratoriossa Suomessamyytävien elintarvikkeiden asetaldehydipitoisuuksia, koska niistä ontoistaiseksi hyvin vähän tietoa.Testien mukaan jogurttien keskimääräinen asetaldehydipitoisuus ylittääriskirajan kahdeksankertaisesti. Vastaavia pitoisuuksia Salaspuro onmitannut useista hedelmämehuista ja soijakastikkeista.– Jopa vauvojen hedelmäsoseista löytyy samoja pitoisuuksia kuinjogurteista. Pitoisuus ylittää aikuisillekin sallitun rajan. Lapsilla painoonsuhteutettu ylitys voi olla jopa 70-kertainen, Salaspuro kertoo.Lisää aiheesta sunnuntain Turun Sanomissa.
  • 50. Alcohol - Cancer - Nrf2/ARE
  • 51. What are free radicals? is any atom or A free radical molecule that has a single unpaired electron in an outer shell.Alcohol/acetaldehyde The free-radical theory of aging (FRTA) states that organisms age because cells accumulate free radical damage over time. For most biological structures, free radical damage is closely associated with oxidative damage. Antioxidan ts are reducing agents, and limit oxidative damage to biological
  • 52. First medicine to combat carcinogenicacetaldehyde L-cysteine + acetaldehyde -- 2-methylthiaszolidine-4-carboxylic acid (L-MTCA) Alcohol dehydrogenase defective
  • 53. Aldehyde Dehydrogenase (ALDH2)Deficiency
  • 54. A tentative study with alcohol/acetaldehyde in A549 cells 1, Cell morphology, 2,Antioxidativ e assay, 3, qRT-PCR 4, Comet Assay
  • 55. Summary 1, MAGE gene family as promissing biomarker need to be further characterized with Estonia cohort, by qRT-PCR, or immunohistochemisty (IHC). 2, For better responses of EGFR and VEGF inhibitors or MAGE vaccines, or for personal medication, genetic assay or gene analysis for NSCLC patients would improve the prognosis. 3, Nrf2, antioxidative, but promoting cancer cell proliferation, and drug resistance , has been regarded as green risk, in cancer chemotherapy. Nrf2 of antioxidation with acetaldehyde may provide some clues for inhibition of proliferation, progression of NSCLC.