MAGE genes and MAGE antigens

1. From metagenes to biomarkers, a possible
approach 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 gene
expression profiling of whole genome) of lung
cancer are immunotherapy target.
2, The prognosis influenced by EGFR and VEGF
mutation and variation.
3, COX-2/Keap1/Nrf2/ARE(s) antioxidant pathways
in lung cancer (NSCLC).
4, Acetaldehyde test in A549, an antioxidant assay
from in vivo to in vitro.

3. 1, Metagenes obtained from GWAS
are 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 and
demethylation of
MAGE genes occur
only in cancer cells
(except testis)
MAGE expression is
known to be activated
by promoter
demethylation.

7. Dendrogram Some of mouse and
representation of the human MAGE gene s
multiple alignment are identical
between MAGE
conserved domains of
human and mouse MAGE
proteins.
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 factors
Barker PA et al: The MAGE proteins: emerging roles in cell cycle progression, apoptosis, and
neurogenetic disease. J Neurosci Res 67:705-712, 2002.
Ohman FK et al: The melanoma antigen genes – any clues to their functions in normal
tissues? Exp Cell Res 265(2): 185-194, 2001.
Chromez, et al 2001, An Overview of the MAGE Gene Family with the Identification of All
Human Members of the Family , Cancer Research

9. Frequent MAGE Mutations in Human Melanoma:
Distribution of mutations in the MAGE genes
Caballero OL et al. Frequent MAGE Mutations in Human Melanoma, 2010 PLOS One

10. Swiss Cohort
Caballero OL et al. Frequent MAGE Mutations in Human Melanoma
, 2010 PLOS One

11. Australia
cohort

12. Global
cohorts by
literatures

13. Chicago cohort
Kufer 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. 2011
Correlation 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 (Melanoma
associated antigen)
participate the
apoptosis!!!
Nasarre P. et al., 2010, Guidance molecules in lung cancer. Cell Adh
Migr. 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 for
personalized melanoma cell vaccine
Table 4 Various combinations of six different TAA distributions in melanoma cell lines
Presence Combination of No. of Presence of Combination of No. of
TAAs expressed TAAs expressed by
of no. of TAAs by melanoma cell cell lines no. of TAAs melanoma cell lines cell lines
(absence of lines (absence of
specific 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 5
5 (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 2
5 (MAGE-1) S-100/Mel-5/HMB-45/Melan-A/ 3 (S-100/Mel-5/Melan-A) MAGE-1/HMB-45/Tyrosinase 1
Tyrosinase 8
5 (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 1
5 (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 1
4 (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 1
4 (S-100/Mel-5) MAGE-1/HMB-45/Melan-A/
Tyrosinase 6
2 (S-100//MAGE-1/Mel-5/HMB-45)Melan-A/Tyrosinase 1
4 (S-100/HMB-45) MAGE-1/Mel-5/Melan-A/Tyrosinase/ 2 2 (S-100/MAGE-1/HMB-45/Melan-A)Mel-5/Tyrosinase 1
4 (MAGE-1/Mel-5) S-100/HMB-45/Melan-A/Tyrosinase 1 1 (S-100/Mel-5/HMB-45/Melan-A/Tyrosinase)MAGE-1 3
4 (MAGE-1/HMB-45) S-100/Mel-5/Melan-A/Tyrosinase 1 1 (S-100/MAGE-1/Mel-5/HMB-45/Melan-A)Tyrosinase 2
4 (Mel-5/HMB-45) S-100/MAGE-1/Melan-A/Tyrosinase 1 1 (S-100/MAGE-1/HMB-45/Melan-A/Tyrosinase)Mel-5 1
Total cell lines 106 0 (S-100/MAGE-1/Mel-5/ HMB-45/Melan-A/Tyrosinase)None 7
TAA, 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 vaccine
MAGE-A3 is a tumor-associated antigen that is not
expressed in most normal cells [31,61]. Approximately 35–
50% of lung cancers express MAGE-A3, and expression has
been associated with poor prognosis [31]. This provided
the rationale for the development of a TCV comprising
MAGE-A3 recombinant protein combined with the AS02B
immunoadjuvant.
In a randomized phase II study, 182 patients with
completely resected, MAGE-A3-expressing, stage IB or II
NSCLC were randomly assigned to receive postoperative
MAGE-A3 vaccine or placebo (2:1) [62]. The HRs for
disease-free interval, disease-free survival (DFS) and overall
survival 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 of
the MAGE-A3-treated patient group. The ongoing phase III
MAGRIT (MAGE-A3 as Adjuvant, Non-Small Cell Lung
Cancer Immunotherapy) trial, initiated in 2007, plans to
enroll approximately 2300 MAGE-A3-expressing patients
with resected stage IB, II, or IIIA NSCLC who will be
randomized to vaccine or placebo, again in the adjuvant
setting. The administration of adjuvant platinumbased
chemotherapy for the treatment of the current NSCLC is
allowed between surgery and randomization. The study is
powered for DFS as the primary endpoint in the total
population and in the cohort without postoperative
chemotherapy [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 EGFR
and VEGF mutation and other genetic
make-up, or variation
Today major personal medication or translational medicine
are from EGFR and VEGF
EGFR and VEGF were not among 599 genes which were down-regulated
and 402 genes which were up-regulated in Urgard E. et all 2011. However, typically the gene
mutation, or methylation, or other tissue-derived molecular information of
EGFR and VEGF has been combined with individual's personal medical history,
family history, and data from imaging, and other laboratory tests for better
effective treatments for a wider variety of conditions.

28. EGFR (Her-1), and VEGF, a clinic
example 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 overexpression
Invassion Alitalo K. et al 2002

32. Gene mutation may increase survival
TKI:
tyrosine
kinase
inhibition,
RR:
response
rate, TTP:
time to
progression

34. VEGF expression associated with tumor growth and survival
(mutation, and silence by methylation)
(A) Overall survival in patients with VEGF-negative and VEGF-positive
tumours (P<0.02).
Fondevila C. et al 2004,
Santini D. et al 2005

35. Summary:
Mutation or gene silence by methylation or
demethylation may help the therapy and
prognosis, so it is good to know the patients’
genetics make-up in clinic.

36. 3, COX-2/Keap1/Nrf2/ARE(s)
antioxidant pathways in lung cancer
(NSCLC)

37. COX-2/Keap1-Nrf2/ARE antioxidant pathway
(Global Keap1 methylation in cancer tissues)
COX-2
The dark side of
Nrf2:
1, Release of Nrf2 EFOX
cause large number
of antioxidant gene
expression, which
make cancer cell
grow faster.
2, Nrf2 enhances
resistance of cancer
cells to
chemotherapeutic
drugs,
Luo C. et al Med. Hypo., 2011

38. Methylation of selected genes
(Method 1)
Kaie Kirotar (UT) provided methylation array
data matrix

39. Method 2: Hypermethylation of Keap1 in
41-79 year old NSCLC patients (A549 (NSCLC) cell
contains several mutations in Keap1 gene)
Kaie Kirotar (UT) provided methylation array data matrix

40. Cell division,
proliferation,
tumorigenesis
EGFR
Nrf2
COX-2 dependent
EFOX on CUL-
Keap1, 2010
Antioxidation

42. 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.

43. 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.

44. 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

45. KEAP1 mutations - dysfunction in
Lung-Tumor-Derived Cell Lines and
Patients

46. Expression of Keap1 and Nrf2 in
NSCLC cell lines
Low Keap1, high Nrf2
NE: nuclear extracts, TP: total protein
Singh A. et al., 2006

47. 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

48. Summary: Internal antioxidants
maintained by Nrf2/ARE
Acetylation
And
deacetylatio
n
SOD

49. 4, Acetaldehyde in A549, an antioxidant
assay from in vivo to in vitro

50. Experimental approaches
TCA cycle
carcinogen
Ethanol is first oxidised to ethanal then ethanoic acid. Note that while different enzymes can be
used in the first step, only aldehyde (ethanal) dehydrogenase (ALDH) is used in the second

51. Jogurteista ja lasten hedelmäsoseista löytyi korkeita
pitoisuuksia asetaldehydiä
Turun Sanomat 7.2 2010 05:00:11
– Tutkin nykyään laboratoriossa, mitä lastenlapseni syövät. Viili on
turvallisempi kuin jogurtit, professori Mikko Salapuro sanoo.
Professori Mikko Salaspuro on huolissaan suomalaisten
elintarvikkeiden asetaldehydipitoisuuksista. Maailman terveysjärjestön
WHO:n alainen kansainvälinen syöväntutkimuslaitos luokitteli
asetaldehydin viime lokakuussa ihmisille syöpää aiheuttavaksi aineeksi
alkoholin juomisen yhteydessä. Se nousi ylimpään riskiluokkaan, johon
kuuluvat myös tupakka ja asbesti.
Suun ja suoliston bakteerit ja hiivat muuttavat alkoholin syöpää
aiheuttavaksi asetaldehydiksi. Ainetta on myös alkoholijuomissa ja
tupakassa.
Lisäksi asetaldehydiä löytyy useista tavallisista elintarvikkeista. Sitä
muodostuu käymisellä valmistettuihin tuotteisiin, kuten
maitovalmisteisiin, soijaruokiin, säilöttyihin vihanneksiin, etikkaan,
kotikaljaan ja simaan.
Asetaldehydiä käytetään myös elintarvikkeiden aromiaineena. Ainetta
lisätään esimerkiksi hedelmämehuihin, virvoitusjuomiin, jälkiruokiin ja
maitovalmisteisiin. Pitoisuuksia ei tarvitse ilmoittaa tuoteselosteessa.
Asetaldehydiä muodostuu myös sisäsyntyisesti joihinkin hedelmiin,
kuten omenoihin, päärynöihin ja marjoihin. Sitä käytetään myös
marjojen ja hedelmien säilöntäaineena.
Mikko Salapuron työryhmä on tutkinut laboratoriossa Suomessa
myytävien elintarvikkeiden asetaldehydipitoisuuksia, koska niistä on
toistaiseksi hyvin vähän tietoa.
Testien mukaan jogurttien keskimääräinen asetaldehydipitoisuus ylittää
riskirajan kahdeksankertaisesti. Vastaavia pitoisuuksia Salaspuro on
mitannut useista hedelmämehuista ja soijakastikkeista.
– Jopa vauvojen hedelmäsoseista löytyy samoja pitoisuuksia kuin
jogurteista. Pitoisuus ylittää aikuisillekin sallitun rajan. Lapsilla painoon
suhteutettu ylitys voi olla jopa 70-kertainen, Salaspuro kertoo.
Lisää aiheesta sunnuntain Turun Sanomissa.

52. Alcohol - Cancer - Nrf2/ARE

53. 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

54. First medicine to combat carcinogenic
acetaldehyde
L-cysteine + acetaldehyde --
2-methylthiaszolidine-4-carboxylic acid (L-MTCA)
Alcohol
dehydrogenase
defective

55. Aldehyde Dehydrogenase (ALDH2)
Deficiency

56. A tentative study with alcohol
/acetaldehyde in A549 cells 1, Cell
morphology,
2,Antioxidativ
e assay,
3, qRT-PCR
4, Comet
Assay

57. 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.