Tumor marker-encoding genes: a bunch of mysterious diamonds in the pile of evolutionary compost

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Tumor marker-encoding genes a bunch of
mysterious diamonds in the pile of evolutionary
compost
A.V. Baranova, George Mason University
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TUMOR MARKERS
Tumor markers are proteins or mRNAs that are
detected in higher-than-normal amounts in the
blood, urine or body tissues of some patients
with certain types of cancer.
Most tumor markers are produced by the tumor
itself, but not by normal tissue
Tumor markers could be immunogenic they could
serve as targets for anti-tumor immune response
3
Changes in tumor marker concentration during the
course of disease
No response to treatment
No treatemnt
Relapse
Second remission
Good response
Remission
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Known examples of tumor markers
PSA and PAPProstate Specific Antigen (PSA) and
Prostatic Acid Phosphatase (PAP) Prostatic
carcinoma, but also in prostatitis and benign
prostatic hyperplasia (BPH)
Alpha-fetoprotein (AFP) AFP is normally produced
by a developing fetus. An elevated level of AFP
strongly suggests the presence of either primary
liver cancer or germ cell cancer
CA125 Ovarian carcinoma. Very good for
monitoring of response and recurrence
Carcinoembryonic Antigen (CEA) Used for
monitoring of colorectal and lung cancer
5
Level of single tumor marker often is not
definitive enough
CANCER
Grey diagnostic area
Benign inflammatory conditions
?
Healthy
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Combining of known tumor markersinto marker
panels will help to increase sensitivity and
specificity of diagnostics
MULTIPLE TUMOR MARKERS NEED TO BE OBTAINED FOR
EACH COMMON TYPE OF CANCER
preferably by high-throughput methods
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Protein based methods of tumor marker discovery

• Occasional findings in course of other research
• Various modifications of SEREX
• (serological expression of cDNA expression
  libraries
• Proteomicsbased methods, including
• 2D PAGE analysis
• SELDI-TOF mass-spectroscopy analysis
• Reverse proteomics arrays

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Aim to reveal the repertoire of
antigens eliciting an antibody response in
cancer patients.
Near 100 of good TMs are revealed by this method
Remove background
True screening
Cancer Immunome Database www2.licr.org/CancerImm
unomeDB
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Two-dimensional polyacrylamide gel
electrophoresis (2D-PAGE) is a technique for
separating complex mixtures of proteins by their
mass and/or charge .
From Wellcome Trust website
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APPLY WOLTAGE TOP TO BOTTOM
From Wellcome Trust website
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APPLY WOLTAGE SIDE TO SIDE
From Wellcome Trust website
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2D PAGE analysis
Normal serum
Tumor serum
Potential tumor marker
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MORE TYPICAL 2D-PAGE GEL
Example from University of Kent website
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Surface-enhanced laser desorption/ionization
(SELDI-TOF) mass-spectrometry
Mass/charge
proteins of interest are selectively absorbed
to a chemically modified surface.
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Normal
Hyperplasia
TUMOR
Normal
Hyperplasia
TUMOR
From Virginia Prostate Center website
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DNA-based Differential Display
A common term for group of methods for a search
of the transcripts which expression in the
sample of interest differs from that in
so-called control sample.
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Its time forcomputational genomics
GenBank contains 5,120,207 of human ESTs on May
30, 2005
In UniGene, individual human ESTs are already
clustered by homology.
Each EST is derived from described tissue source
including tumor and normal samples.
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Example of ESTs that belong to the same
gene/UNIGENE cluster
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Manual verification of cDNA libraries sources
cDNA libraries in GeneBank
TUMOR
NORMAL
UNDEFINED
A sources for cross-verification UNIGENE, CGAP,
TIGR, STRATAGENE, PubMed
Figure of year 2001
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HSAnalyst and ClustOut software
HSAnalyst and ClustOut software developed in
Functional Genomics Group, IGG RAS
Software is able to sort EST clusters according
to their size, tissue origin or other categories
describing individual ESTs
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First round of work (year 2000). Cluster-sorting
software HSAnalyst
To list all human clusters containing 100 of
ESTs from tumor-derived cDNA libraries with more
than 16 ESTs in the cluster - 21 cluster
To list all human clusters containing less than
10 of ESTs from normal cDNA libraries with more
than 16 ESTs in the cluster 83 clusters
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Statistics
FEBS Letters, 508 (2001), 143-148
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RT-PCR of Brachiury gene in human tumor and
normal tissues
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Cytokine production and cytotoxic activity of
CTLs specific for three Brachyury-derived
peptides. A, CD8 Tcells generated from PBMC of
a healthy donor against peptidesT-p2 and T-p3
were stimulated for 24 h in the presence of
Brachyury (T)-specific peptides or irrelevant
peptide-pulsed autologous DCs. IFN-g was
evaluated in the supernatants by ELISA.
B. Cytotoxic activity (6-h assay) of CTLs
generated with peptideT-p2 against peptide-pulsed
C1R-A2 targets. Two effector-to-target ratios
(ET) were used as indicated.
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Cytotoxic activity of Brachyury-specific CTLs
against tumor targets. CTLs established from the
blood of (C) a colorectal cancer patient
(patient1) and (D) an ovarian cancer patient
(patient 2) were used after three IVS for
cytotoxic killing of H441 and AsPC-1 tumorcells.
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Tumor marker properties of Brachiury were
predicted computationally

• It was the first study to show that (a) a T-box
  transcription factor and (b) a molecule
  implicated in mesodermal development, i.e., EMT,
  can be a potential target for human
  T-cellmediated cancer immunotherapy.
• up-regulation of Brachyury occurs in certain
  tumor tissues and cancer cell lines
• Brachyury-specific CTLs can be generated from the
  blood of cancer patients and normal donors,
  which, in turn, can effectively lyse
  Brachyury-expressing tumor cells.

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Recent round of work (year 2006).
Cluster-sorting software ClustOut
Clusters containing 100 ESTs from human
libraries derived from tumor samples - 328
clusters
Clusters containing less than 10 ESTs from human
libraries derived from normal samples - 501
clusters
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Tumor prevalent clusters
501
Unknown human genes with unstudied ORFs
52 are genes with Gene IDs (10)
13
10 known tumor markers
Non-coding mRNAs
Insulin 268/3113
466 (87)
Parathyroid hormone 1/83
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Known tumor markers in cancer-specific subtraction
10/501
Gene Function Normal/cancer
MYEOV myeloma overexpressed gene 2/113
MAGEA6 melanoma antigens, family A 8/103
MAGEA3 melanoma antigens, family A 7/76
MAGEA12 melanoma antigens, family A 0/27
MAGEA1 melanoma antigens, family A 1/13
MAGEA9 melanoma antigens, family A 1/13
CEACAM8 carcinoembryonic antigen-related cell adhesion molecule 8 2/30
CTAG2 Cancer/testis antigen 2 2/20
TRAG3 taxol resistance associated gene 3 0/10
APOBEC1 apolipoprotein B mRNA editing enzyme, catalytic polypeptide 0/59
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Prospective marker proteins in cancer-specific
subtraction
Gene Function Norm/cancer
ASCL1 achaete-scute complex-like 1 (Drosophila) 33/394
WNT7B wingless-type MMTV integration site family, member 7B 22/285
DLL3 delta-like 3 (Drosophila) 18/193
NOX1 NADPH oxidase 1 4/76
KREMEN2 kringle containing transmembrane protein 2 5/74
SPRR2A small proline-rich protein 2A 1/69
MS4A3 membrane-spanning 4-domains, subfamily A, member 3 5/65
GPR35 G protein-coupled receptor 35 3/46
SPRR1A small proline-rich protein 1A 4/46
OTP orthopedia homolog (Drosophila) 1/41
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Known human genes represented by only
tumor-derived EST
Published in FEBS Letters, Nov 2001
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Reverse subtraction genes prevalent in normal
samples
Tumor prevalent clusters
501 clusters with less than 10 of ESTs from
normal tussues
328 with 100
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Normal prevalent clusters
1048
Unknown human genes with unstudied ORFs
612 are genes with Gene IDs (56,6)
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Non-coding mRNAs
10 crystallins
399 (36,9)
7 keratins
Tear prealbumin, lens intrinsic membrane protein
2
16 (!) different solute carrier proteins (2,6)
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Non-coding RNAs in tumor-specific and
normalspecific subtractions

• In normal prevalent clusters
• 399 out of 1048 (36,9)
• In tumor prevalent clusters
• 466 out of 501 (87)

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Possible explanation

• Tumors express more non-coding mRNA than normal
  tissue due to
• -- overall weakness of gene expression
  control
• (probably due to demethylation of genome)
• Awakening of dormant promoters

Expression of sequences adjacent to dormant
promoters with splicing according to cryptic
splice sites
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• Experimental study
• of Tumor Specificity
• Whether software based predictions are true or
  not???
• Kozlov et al.,
  2003
• 
  Krukovskaja et al., 2004

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Hs.133107
- Non-coding mRNA - Located on Chromosome 12p13 -
Represented by 20 EST from the 5 tumor- derived
libraries from Brain Tumors, Lung Carcinomas,
Ovarian Carcinomas
20/20
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Amplification of Hs.133107 Fragment on Human
Tumor MTC Panel (Clontech)
1 2 3 4 5 6 7 8 9
NC PC
344 bp
1 100bp DNA ladder 2 Breast Carcinoma 3
Lung Carcinoma 4 Colon Adenocarcinoma 5 Lung
Carcinoma
6 Prostatic Adenocarcinoma 7 Colon
Adenocarcinoma 8 Ovarian Carcinoma 9
Pancreatic Adenocarcinoma PC - Human DNA
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Amplification of Hs.133107 Fragment on Human
NORMAL MTC Panel 1 (Clontech)
1 2 3 4 5 6 7 8 9 NC
PC
344 bp
1 100 bp DNA ladder 2 Brain 3 Heart 4
Kidney 5 - Liver
6 Lung 7 Pancreas 8 Placenta 9 Skeletal
Muscle PC - Human DNA
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Amplification of Hs.133107 Fragment on Human
NORMAL MTC Panel 2 (Clontech)
1 2 3 4 5 6 7 8 9 NC
PC
344 bp
1 100bp DNA ladder 2 Colon 3 Ovary 4
Peripheral Blood Leukocyte 5 - Prostate
6 Small Intestine 7 Spleen 8 Testis 9
Thymus PC - Human DNA
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Amplification of Hs.133107 Fragment on Human
Immune System MTC Panel, (Clontech)
1 2 3 4 5 6 7 8 NC
PC
344 bp
1 100 bp DNA ladder 2 Bone Marrow 3 Fetal
Liver 4 Lymph Node 5 Peripheral Blood
Leukocyte
6 Spleen 7 Thymus 8 Tonsil PC - Human DNA
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Amplification of Hs.133107 Fragment onHuman
Fetal MTC Panel (Clontech)
1 2 3 4 5 6 7 8 9 NC PC

344 bp
1 100 bp DNA ladder 2 Brain 3 Heart 4
Kidney 5 - Liver
6 Lung 7 Skeletal Muscle 8 Spleen 9
Thymus PC - Human DNA
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Conclusion Hs.133107 is a Tumor-Specific
Sequence
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Hs.154173
- Non coding mRNA - Located in the intergenic
spacer region within rRNA encoding unit -
characteristic for lung carcinoma, testicular
teratocarcinoma
23/23
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Amplification of Hs.154173 Fragment on Human
Tumor MTC Panel (Clontech)
1 2 3 4 5 6 7 8 9 NC PC
443 bp
1 100bp DNA ladder 2 Breast Carcinoma 3
Lung Carcinoma 4 Colon Adenocarcinoma 5 Lung
Carcinoma
6 Prostatic Adenocarcinoma 7 Colon
Adenocarcinoma 8 Ovarian Carcinoma 9
Pancreatic Adenocarcinoma PC - Human DNA
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Amplification of Hs.154173 Fragment on Human
Normal MTC Panel 1 (Clontech)
1 2 3 4 5 6 7 8 9 NC
PC
443 bp
1 100 bp DNA ladder 2 Brain 3 Heart 4
Kidney 5 - Liver
6 Lung 7 Pancreas 8 Placenta 9 Skeletal
Muscle PC - Human DNA
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• Three other normal panels looks same as previous
  
• Conclusion
• Hs.154173 is
• a Tumor-Specific Sequence

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Hs.133294

• Protein encoding mRNA
• Located on Chromosome 1q21
• IQGAP3 gene
• Weakly similar to IQGAP (human RAS
  GTPase-activating-like protein IQGAP1)
• Represented in prostate tumor, HNSCC, breast
  carcinoma, oligodendroglioma, colon carcinoma,
  CML, lung carcinoma, ovarian carcinoma, uterus
  carcinoma, adrenal adenoma
• Minor occurrences in normal testis and germinal
  B-cells

58/62
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mRNA isoforms of IQGAP3 gene
5905
5336
Only 3 underspliced mRNA variant of IQGAP3 gene
is tumor specific (this part of the gene is
non-coding either)
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Conclusion One of the mRNA isoforms of the
IQGAP3 gene represented by Hs.133294 is a
Tumor-Specific Sequence
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Back to fundamental aspect of the tumor-specific
mRNAs
WHAT IS THEIR FUNCTION ???
What is a degree of conservativeness between
human tumor-specific genes and mouse genes?
Do they have orthologs in the mouse genome?
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Closer look into well-established tumor-markers

• 1) For most of the known tumor markers no clear
  function in the adult human cells is shown
• 2) Looks like these genes not expressed in normal
  tissues just because they not needed there
• 3) Looks like these genes are expressed in tumors
  just because tumors dont care !!!

Prediction no clear function ? low level of
conservation
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VISTA special visualization tool for distant
cross-species comparison
http//www-gsd.lbl.gov/vista/
Inna Dubchak and Eugene Rubin, 2002
Lawrence Berkeley National Laboratory (LBNL)
This tool allow graphical representation of the
alignment plots
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How normal gene looks in VISTA
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Cclbr Alignment 1 Seqs human/mouse Criteria
75, 50 bp Regions 31 X-axis human Resolution
15 Window size 50 bp
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How tumor marker gene looks in VISTA (MAGE A4)
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MGEA4 tumor marker
meningioma expressed antigen
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You dont believe me? Here is MAGE A9 another
one.
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More detailed VISTA view
NRAS - oncogene
FUGU and CHICKEN ARE PRESENT !!!!
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More detailed VISTA view
CEACAM6
Nothing here
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Some genes are right in the middle some exons
are conservative, some are not (non-coding ones)
RFP2
Here it is
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Graphical alignments
NRAS
RFP2
CEACAM6
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• What about our own flock
• of predicted tumor markers ???

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SIMPLE BLAST analysis
74 Clusters corresponding to mouse genes gt80 -
nt gt60 -aa
170 cancer strictly cancer specific EST clusters
83 Clusters not corresponding to mouse genes
(evolutionary new) lt 70 nt lt40 - aa
13 borderline clusters
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Evolution often goes by duplications
What is the genomic source of these
evolutionary new tumor markers????
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22 of 170 cancer specific gene clusters are
recently (only in primates) duplicated in human
genome !!!
Including known tumor marker genes
MAGE A4 MAGE A9 CTAG2 (cancer/testis specific
antigen 2)
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IDEA

• In course of evolution new sequences
• become ready to be expressed
• as they got joined to dormant promoter
• But unnecessary promotors are thoroughly
  suppressed in well-differentiated adult cells
• In tumors all these yet-to-be genes
• got their chance

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Same is true for parts of the genes separate
exons, especially non-coding exons
cancer specific alternative splicing
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Something to think about

• 1. Can we use these non-coding tumor-specific
  mRNA as a tumor markers and targets for
  immunotherapy?
• (protein-coding genes could be used more readily)
• Yes.
• They always encode short ORFs linked with AUGs.
  Such short ORFs are TRUE non-self antigens.

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Something to think about

• 2. Why known tumor markers have
  carcino-embryonic or carcino-testicular profiles
  of expression?
• -- embryos contain lots of less differentiated
  cells with relaxed expression control
• -- testes are covered by blood-testes barrier, so
  immune system is not watching for an expression
  of non-self ORFs
• It explains
• broadest pattern of expression observed in tested

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George Mason University, Fairfax, VA, USA

• A.Baranova, M.Sikaroodi, G,Manyam, P. Gillevet

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The Biomedical Center, St.Petersburg, Russia
A.P. Kozlov, L.L.Krukovskaja, D.Polev, I.
Duhovlinov, Yu.Galachjants, N.Samusik
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Vavilov Institute of General Genetics, Moscow,
RussiaT.TyazhelovaNCI, NIH

C.Palena, J.Schlom S.OBrien
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Experimental example DLEU2 gene in human and
mouse (evol new gene)
Red mouse-specific exons only Green human
specific exons only
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