PhD Defense

A study on the Genetic Alterations of the Signal
Transducing Molecules and their role in the
development and progression of Colorectal Cancer in
Kashmir Valley
Aga Syed Sameer
PhD/Bio/019
Co-Supervisor
Dr. Zafar Amin Shah
Additional Professor
Department of Immunology &
Molecular Medicine
Co-Supervisor
Dr. Safiya Abdullah
Associate Professor
Department of Immunology &
Molecular Medicine
Co-Supervisor
Dr. Nisar A Chowdri
Professor
Department of General Surgery
Supervisor
Dr. Mushtaq A Siddiqi
Professor & Head,
Department of Immunology & Molecular Medicine

Hanahan D and Weinberg RA. The hallmarks of cancer. Cell. 2000; 100:57-70.
INTRODUCTION
Cancer
Aga Syed Same
Cancer is a clonal disorder.
It is composed of malignant
cells of several distinguishable
characteristics such as:
• immortality,
• faster growth,
• unable to establish cell-cell
interaction,
• propensity to invade,
metastasize and
• grow in an abnormal cellular
environment.

High incidence rates are found in
western world populations, i.e.
Western Europe, North America, and
Australia.
Jemal et al., Global cancer statistics. CA Cancer J. Clin. 2011; 61: 69-90.
INTRODUCTION Colorectal Cancer
Aga Syed Same
Colorectal Cancer is the third most common cause of cancer-
related death in the western world.
Colorectal cancer is the Third most common cancer in men and
the Second most common cancer in women worldwide.
The annual incidence of CRC worldwide has been estimated to be
at least half a million. It is a commonly diagnosed cancer in both
men and women.
The lowest rates of CRC are found in
the sub-Saharan Africa, South
America and Asia.

Jemal et al., Global cancer statistics. CA Cancer J. Clin. 2011; 61: 69-90.
INTRODUCTION
Worldwide
Aga Syed Same

Center MM et al., Worldwide Variations in Colorectal Cancer. CA Cancer J Clin 2009;59;366-378
INTRODUCTION Worldwide
The majority of registries with the highest incidence rates of colorectal
cancer were located in Europe, North America, and Oceania.
In contrast, the lowest rates were observed from registries in Asia,
Africa, and South America.

INTRODUCTION Developing Nations
Among both males and females, the lowest rates of colorectal cancer incidence
were observed for registries in India (Nagpur, Poona and Karunagappally), Oman,
Egypt (Gharbiah), Algeria (Setif), and Pakistan (South Karachi).
In these economically developing regions of the world, low CRC incidence rates
may reflect a lower prevalence of known risk factors.

Sameer AS et al., SMAD4 - Molecular gladiator of the TGF-β signaling is trampled upon by
mutational insufficiency in Colorectal Carcinoma of Kashmiri population: An analysis with relation
to KRAS proto-oncogene. BMC Cancer. 2010. 10:300.
Javid G et al., Incidence of colorectal cancer in Kashmir valley, India. Indian J Gastroenterol. 2011
Feb 12.
INTRODUCTION India & Kashmir
Aga Syed Same
In India, CRC incidence rates vary markedly, with rates per
100,000 among males in the time period 1998–2002 reported
to to be 4.1 & 3.6 among females (Karunagappally).
The scenario in the Kashmir valley is not different from that of
the world. Representing 4th most common cancer in Kashmir.
In Kashmir too, it is the most prevalent form of cancer after
esophageal and gastric.

Risk Factors
CRC
IBD
FOOD
AGE
ETHINICITY
OBESITY
INHERITED
SYNDROMES
PHYSICAL
INACTIVITY
SMOKING
ALCOHOL
INTRODUCTION
Weitz J et al., Colorectal cancer . Lancet 2005; 365: 153–65
INFECTION

Risk Factors
Weitz J et al., Colorectal cancer . Lancet 2005; 365: 153–65
INTRODUCTION

Molecules involved in
Colorectal Cancer Pathogenesis
Normal
Small
Adenoma
Large
Adenoma
Cancer
Genetic Instability
Metastasi
s
Vogelstein B et al., Genetic alterations during colorectal-tumor development. N Engl J Med. 1988;
319: 525-532.
Fearon ER and Vogelstein B. A genetic model for colorectal tumorigenesis. Cell. 1990; 61:759-767.
INTRODUCTION
Aga Syed Same

Molecules involved in
INTRODUCTION
Narayan S and Roy D. Role of APC and DNA mismatch repair genes in the development of
colorectal cancers. Mol. Cancer 2003, 2:41

Wnt Pathway
The dynamics of β-catenin
dependent Wnt signalling pathway.
Wnt signalling has been shown to
regulate cell fate decisions in
development and to affect cell
proliferation, morphology, migration,
apoptosis, or differentiation in a variety
of tissue settings.
Recent reports also indicate that Wnt
signalling promotes stem cell self-
renewal in certain tissues
INTRODUCTION
Polakis P. Wnt signaling and cancer. Genes Dev. 2000; 14:1837-51.

Mutation Prone Molecules In Wnt PathwayINTRODUCTION
Functional domains and Mutational spectrum of APC protein.
A compilation of germline and somatic mutations in APC illustrates
selection for mutations in the mutation cluster region (MCR). MCR
mutations result in truncated proteins retaining β-catenin binding but not
regulatory activity. Somatic MCR mutations are more frequently selected
for in FAP patients with germline mutations outside of the MCR.
Fearnhead et al. The ABC of APC. Hum Mol Genet. 2001; 10:721-733

Mutation Prone Molecules In Wnt PathwayINTRODUCTION
Functional domains of β-catenin protein.
The central part is made up of 12 highly homologous armadillo repeats
(boxes 1–12) which mediate most interactions with other proteins. Serine
and threonine residues 33, 37 and 41 (insert) are the GSK-3
phosphorylation sites. Serine 45 is the target of priming phosphorylation
by CKI. Mutation of one of these residues prevents degradation of ß-
catenin.
Fearnhead et al. The ABC of APC. Hum Mol Genet. 2001; 10:721-733

 This illustration of the Ras signaling pathway highlights proteins affected
by
mutations in developmental disorders and cancer. (Shown in Blue)
 Mitogen-activated protein kinase (MAPK) cascades are among the most
thoroughly studied of signal transduction systems and have been shown
to participate in a diverse array of cellular programs, including cell
differentiation, cell movement, cell division, and cell death (Apoptosis).
MAP Kinase Pathway
INTRODUCTION
Pearson G et al. Mitogen-activated protein (MAP) kinase pathways: regulation and physiological
functions. Endocr Rev. 2001; 22:153-83.

TGF β PathwayINTRODUCTION
Schematic representation of SMAD dependent TGF-β Pathway.
TGF-β acts as a tumor
suppressor by inhibiting cellular
proliferation or by promoting
cellular differentiation or
apoptosis). In the initial stages
of tumorigenesis, a cell loses its
TGF-β mediated growth
inhibition as a result of mutation
or loss of expression of the
genes for one or more
components of the TGF-β
signalling pathway.
Massague J. TGF-β signal transduction. Annu Rev Biochem 1998, 67:753–791.

TGF β PathwayINTRODUCTION
The Smad family. Diagramatic
representation of structure of
three subfamilies of Smads.
A. General structure of Smads B. R-Smads (Smad1, Smad2, Smad3, Smad5 and Smad8) C. Co-Smads (Smad4)
D. I-Smads (Smad6 and Smad7)
The protein diagrams are arbitrarily aligned relative to their C-termini. The MH1 domain is coloured in light green
and the MH2 domain in magenta. Selected domains and sequence motifs are indicated as follows: a-helix H2, L3
and H3/4 loops, b-hairpin, the unique exon 3 of Smad2 (ex3), NLS and NES motifs or putative (?) such motifs, the
proline-tyrosine (PY) motif of the linker that is recognised by the Hect domain of Smurfs, the unique SAD domain
of Smad4 and the SSXS motif of R-Smads with asterisks indicating the phosphorylated serine residues.
Smad4 gene mutation map.
Black arrows indicate nonsense, frameshift and inframe deletion mutations, and white
arrows indicate missense mutations. Red Lines show exons (Mutational Cluster
Region) harbouring higher mutation rate.
Attisano L et al. The Smads. Genome Biology. 2001; 2: 3010.1–8.

The TP53 pathway.
The TP53 pathway is complex. At
least 50 different enzymes can
covalently modify p53 to alter its
stability, cellular location or
activity17.
The TP53 pathway is composed of
a network of genes and their
products that are targeted to
respond to a variety of intrinsic and
extrinsic stress signals that impact
upon cellular homeostatic
mechanisms that monitor DNA
replication, chromosome
segregation and cell division
TP53 PathwayINTRODUCTION
TP53 single
nucleotide
polymorphisms:
locations in the p53
protein and DNA
sequences.
Levine AJ et al. The P53 pathway: what questions remain to be explored? Cell Death Differ. 2006;

Pathways involved in
RATIONALEOFSTUDY
Fearon ER and Vogelstein B. A genetic model for colorectal tumorigenesis. Cell. 1990; 61:759-767.
Aga Syed Same

1
• Literature and studies around the globe
were searched
2
• Important signal transduction molecules -
of these four pathways involved in the
CRC pathogenesis were identified
3
• Mutational cluster regions and/or hotspot
in each gene was identified
RATIONALEOFSTUDY

RATIONALEOFSTUDY
Aga Syed Same
• APC (MCR)
• B-catenin (Exon 3)
Wnt
Pathway
• KRAS (Codon 12, 13)
• Braf (Exon 15)
MAP
Kinase
• SMAD4 (MCR)
TGF
Beta
• Polymorphism –
Codon 47 & 72TP53

Power of StudyRATIONALEOFSTUDY
To attain 80%
Size of samples was found to
be 67

Identification of patients with CRC
• Characterization of CRC patients into familial &/or
sporadic cases
• Gathering case history about their life style
• Collection of affected tissue & blood samples (as
control)
Screening of the CRC patient
• Analysis for genetic aberrations in Signal
Transducing Molecules
• Identification of the risk factors to which our
population is exposed
Mutational Profiling of CRC cases
• Correlation of Signal Transducing Molecules’ gene
mutation with CRC development
• Development of genetic model for each gene CRC in
Kashmir valley – emphasizing aberration frequency
AIMSOFSTUDY
Aga Syed Same

Samples
• Patients were recruited from General Surgery
• Patients who were being treated for Colorectal Cancer (CRC) for the first
time were recruited
Size
• Surgical CRC specimens (n=86)
• Matched normal tissue from a distant site
• Blood samples also collected from patients
• Blood samples were collected from control (n=160)
Interview
• A pretested, semi-structured questionnaire was used to collect
information from each selected patient
• Clinico-pathological parameters, including age, gender, site, dukes
stage and histopathological grade
METHODOLOGY
Aga Syed Same

METHODOLOGY
Aga Syed Same
DNA
Extraction
• Isolated from blood and tissue samples by
phenol/chloroform or ammonium acetate
extraction method
PCR-SSCP
Analysis
• Of the PCR products was carried out on a 6%
non-denaturing polyacrylamide gel utilizing
either non-radioactive silver staining
DNA
sequencing
• Using the ABI prism 310 automated DNA
sequencer

MS PCR
• Methylation Specific PCR analysis was done to for APC 1A and
1B promoter
PCR RFLP
For
Methylation
• PCR- RFLP analysis was done to for SMAD4 promoter using
MspI/HpaII digestion method for methylation
METHODOLOGY
Aga Syed Same
PCR RFLP
For Mutation
• PCR- RFLP analysis was done for B-catenin to detect mutations
in codon 32/33
PCR RFLP
For TP53
Polymorphis
m
• PCR- RFLP analysis was done for TP53 to determine genotypes
of codon 47 & 72

METHODOLOGY
Aga Syed Same
Statistical
analysis
• Was done by using PASW version 18 software
• Fisher’s exact test for homogeneity of proportions was
used to determine the significance of the mutation pattern
Correlation
• The OR/Pvalue was used to determine the association of
identified mutations with various clinico-epidemiological
characteristics such as age, lymph node/s involved, clinical
tumor stage and histopathological grade of the tumor
P-Value
• Statistical significance was considered when p ≤ 0.05

Frequency distribution analysis of selected demographic and risk factors in colorectal
cancer cases and controls.
Variable Cases
(n=86)
Controls
(n=160)
P Value
Age group
≤50
>50
30 (34.9%)
56 (65.1%)
56 (35.0%)
104 (65.0%)
1
Gender
Female
Male
37 (43.0%)
49 (67.0%)
72 (45.0%)
88 (55.0%)
0.76
Dwelling
Rural
Urban
59 (68.6%)
27 (31.4%)
104 (65.0%)
56 (35.0%)
0.56
Smoking status
Never
Ever
31 (36.0%)
55 (64.0%)
75 (46.8%)
85 (53.2%)
0.10
Pesticide Exposure
Never
Ever
33 (38.4%)
53 (61.6%)
75 (46.8%)
85 (53.2%)
0.20
Cases & Controls
RESULTS

A)Image showing the entire colon
of the third generation FAP
patient, which was removed by
APR surgery. Image showing the
inner lining of the colon, notice
hundreds of polyps al over the
surface of the colon and a rosette
shaped malignant tumor at
transverse section of the colon.
B) A closeup of the rosette
shaped malignancy of the colon.
The tumor was classified as
T4N2M1, Histopathology
revealed the tumor to be poorly
differentiated Adenocarcinoma.
Sameer AS et al. A rare case of FAP in Kashmiri population. Indian J of Surgery. 2011.
RESULTS

DNA Extracted from samples
Agarose gel electrophoresis of DNA isolated from blood, tumor tissue,
and adjacent normal tissue of colorectal cancer patient.
Lane M: Lambda DNA Eco RI and Hind III digest
Lane 1, 3 and 6: DNA from CRC tissue
Lane 2, 4 and 5: DNA from Control Blood
RESULTS

Mutation Spectrum of Signal
RESULTS

APC MCR Amplicons
Representative gel picture of MCR regions of APC gene comprising of Exon 15
APCA (295bp); APCB (293bp); APCC (290bp); APCD (295bp) and APC Full (890bp)
Lane M: Molecular size marker 100bp (Middle Prominent Band =500bp)
Lane 1-6: Amplified product from cancer samples
RESULTS-APC

The overall mutation rate of mutation cluster region (MCR) of APC gene
among 86 patients was found to be 12.8% (11 of 86).
Four missense mutations, three nonsense mutations and four frameshift
mutations - including three deletions and one insertion.
Among the three nonsense mutations; two were Leu> Stop and one was
Lys>Stop.
We also found a novel SNP in our study, G>A polymorphism in codon
1492 of APC gene. The polymorphism changes ACG to ACA, without
changing the resulting amino acid residue.
We found, that among 86 CRC cases, only 14 (16.3%) had homozygous
wild (GG) genotype while as 53 (61.6%) had homozygous variant (AA)
genotype and 19 (22.1%) had heterozygous (GA) genotype
Aga Syed Same
RESULTS-APC

Codon 1492 Status of APC
Pie chart showing the frequency distribution of three different variants of
codon 1492 of APC gene in 86 CRC patients
Aga Syed Same
RESULTS-APC

Polymorphism of codon 1492
Polymorphism of codon 1492 in
MCR region of APC gene.
•Homozygous ACG (Wild)
•Heterozygous ACN
•Homozygous ACA (Variant)
Aga Syed Same
RESULTS-APC

Sequence Results of APC
Partial electropherograms representing the
mutant (Above) and normal (Below) {Shown by
arrows}, of MCR region of APC gene.
•Deletion of AAAAG pentamer at codons 1307/08/09
in MCR region of APC gene
•Deletion of AA dimer at codons 1307/08 in MCR
region of APC gene
Partial electropherograms representing the
mutant (Above) and normal (Below) {Shown
by arrows}, of MCR region of APC gene.
Insertion of A at codon 1525 in MCR region of
APC gene
RESULTS-APC

β-catenin amplicons
Representative gel picture β-catenin amplicons; β-catenin Exon 3 (200bp)
RESULTS-β-catenin

Representative gel picture of PCR-RFLP (HinfI digest) analysis of 200bp amplicon of
exon 3 of β-catenin gene
Lane M: Molecular size marker 50bp (Two Prominent bands = 250bp & 500bp)
Lane 1-14: HinfI digested amplicons from cancer samples; Lane 11 shows mutant β-
catenin codon 32.
RESULTS-β-catenin
β-catenin RFLP analysis

The overall mutations rate of β-catenin gene was found to be
8.1% (7 of 86).
Out of these 7 mutations, three affected codon 32, three
affected codon 49 and one affected codon 45.
Five of the seven mutations were missense and two were
nonsense.
Both nonsense mutations affected codon 49 changing lysine to
stop codon leading to truncation of protein.
Nature of β-catenin in 7 colorectal carcinoma patients from Kashmir valley
Patient ID Mutation Amino Acid
Change
Affected Codon Effect
X1 GAC>GGC Asp>Gly 32 MS
X5 AAA>TAA Lys>Stop 49 NS
X9 AAA>TTA Lys>Leu 49 MS
X62 AAA> TAA Lys>Stop 49 NS
X82 TCT>TTT Ser>Phe 45 MS
Aga Syed Same
RESULTS-β-catenin

KRAS & Braf amplicons
Representative gel picture of KRAS and Braf amplicons.
KRAS Exon 1 (162bp); Braf Exon 15 (223bp)
RESULTS-KRAS

SSCP Analysis of KRAS
Representative gel picture of SSCP analysis showing mobility in KRAS exon 1 amplicon
of cancer tissue DNA against amplicon of normal tissue DNA
Lane 6 corresponding to C3 sample shows the mobility shift indicating some aberration present in the
amplicon
RESULTS-KRAS

The overall mutation rate of KRAS gene was found to be 24.4% (21 of
86) CRC cases. These 21 mutants exhibited 22 mutations in total.
In 52.4% (11/21) of cases mutations occurred in codon 12 only, 23.8%
(5/21) in codon 13 only while as one tumor (X04) had mutations in both
12 & 13 codons.
In addition, we also found two types of new novel mutations in KRAS
oncogene which had not been reported yet.
One was the A:T>T:A transversion of in codon 16 resulting in the
truncation (AAG>TAG; Lys>Stop) of Kras protein in two (2/21; 9.5%)
cases.
Second was the C:T>T:C transition at codon 19 leading to change of
leucine to phenaylalanine (CTT>TTT) in two (2/21;9.5%) tumor tissues
Aga Syed Same
RESULTS-KRAS

Statistical analysis of the various clinicopathological variables revealed a
significant association (p < 0.05) between the KRAS mutation and the
age and tumor location.
The DNA sequence analysis did not reveal any Braf gene mutations in
any one of the 86 CRC cases contrary to our expectations
Variable Total
N=86
Mutants
M=21 (24.4%)
P value*
Age group
≤60
>60
52 (60.5%)
34 (39.5%)
17 (%)
4 (%)
0.03
Tumor location
Colon
Rectum
36 (41.9%)
50 (58.1%)
13(%)
8 (%)
0.04
Aga Syed Same
Seven cases were G12D (GGT > GAT), three cases G13D (GGC
> GAC), two cases G12S (GGT > AGT), two cases G12A (GGT
>GCT), two cases G13R (GGC>CGC), two cases K16X
(AAG>TAG), two cases L19F (CTT>TTT), one case G12C (GGT>
TGT) and one case G13C (GGC>TGC).
RESULTS-KRAS

Sequence Results of KRAS
Partial nucleotide sequence (reverse) representing the normal (Below) and mutant (Above)
{Shown by asterik and arrows}, of KRAS exon 1 gene.
A: Transition of C>T at codon 19; CTT>TTT; Lue>Phe in Exon1 of KRAS gene.
B: Transversion of A>T at codon 16; AAG>TAG; Lys>Stop in Exon1 of KRAS gene.
Aga Syed Same
RESULTS-KRAS

Sequence Results of KRAS
Partial nucleotide sequence (reverse) representing the normal (Below) and mutant (Above)
{Shown by asterik and arrows}, of KRAS exon 1 gene.
A: Transition of G>A at codon 12; GGT>GAT; Gly>Asp in Exon1 of KRAS gene.
B: Transversion of G>T at codon 12; GGC>TGC; Gly>Cys in Exon1 of KRAS gene.
C: Transition of G>C at codon 12; GGT>GCT; Gly>Ala in Exon1 of KRAS gene.
Aga Syed Same
RESULTS-KRAS

SMAD4 MCR amplicons
Representative gel picture of MCR regions of SMAD4 gene comprising of Exon2, 8, 9, 10 and 11.
Exon2 (175bp); Exon 8 (264bp); Exon 9 (332bp); Exon 10 (213bp); Exon 11 (299bp)
RESULTS-SMAD4

The overall mutation rate of mutation cluster region (MCR) of
SMAD4 gene among 86 patients was found to be 18.6% (16 of
86). Analysis of the mutation spectrum of MCR region of SMAD4
gene revealed 16 mutations in total.
The frame shift mutation was observed in codons 415/416 (exon
9) due to deletion of AGACA pentamer.
There were ten missense mutations, two nonsense mutations,
three silent mutations and one frameshift mutations.
The two nonsense mutations included, CAG>TAG transition
leading to Gln>Stop at codon 442 and other CGA>TGA transition
leading to Arg>Stop at codon 445, both occurred in exon 10 of
SMAD4 gene.
Aga Syed Same
RESULTS-SMAD4

Among the 16 mutations seen, there were one (6.25%) in
exon 2, six (37.5%) in exon 8, three (18.75%) each in exon
9, exon 10, and exon 11 of MCR region of SMAD4 gene.
Clearly the hot spot exon within the MCR region of the
SMAD4 gene was exon 8 and within the exon 8 it was the
codon 361 which was frequently aberrated due to
mutations.
Furthermore, we found a significant association of Tumor
location, Nodal status and Bleeding PR/Constipation with
the mutation status of the SMAD4 gene (P = < 0.05).
There was also a significant association of the SMAD4
mutants with KRAS mutants.
Aga Syed Same
RESULTS-SMAD4

Clinico-epidemiological variables of the 86 colorectal carcinoma patients versus 16
mutant phenotypes of SMAD4 gene.
Variable Total
N=86
Mutants
M=16 (18.6%)
P value*
Tumor location
Colon
Rectum
36 (41.9%)
50 (58.1%)
12 (33.3%)
4 (8.0%)
< 0.01
Nodal status
Involved
Not Involved
48 (55.8%)
38 (44.2%)
14 (29.2%)
2 (5.3%)
< 0.01
Tumor grade
A+B
C+D
38 (44.2%)
48 (55.8%)
2 (5.3%)
14 (29.2%)
< 0.01
Bleeding
PR/Constipation
Yes
No
60 (69.8%)
26 (30.2%)
15 (25.0%)
1 (3.8%)
< 0.01
Pesticide exposure
Ever
Never
53 (61.6%)
33 (38.4%)
13 (24.5%)
3 (9.1%)
< 0.05
KRAS Status
Wild-type
Mutated
65 (75.6%)
21 (24.4%)
5 (7.7%)
11 (52.4%)
< 0.01
*Pearson’s two proportion test
Aga Syed Same
RESULTS-SMAD4

Sequence Results of SMAD4
Partial nucleotide sequence representing the mutant (Above) and normal (Below) {Shown by asterik
and arrows}, of MCR region of SMAD4 gene.
A: Transition of G>A at codon 340; AAG>AAA; Arg>Lys in Exon8 of SMAD4 gene
B: Transition of G>A at codon 361; CGC>CAC; Arg>His in Exon8 of SMAD4 gene
C: Transversion of T>G at codon 362; TTT>TTG; Phe>Leu in Exon8 of SMAD4 gene
D: Transition of G>A at Codon 415; AGA>AAA; Arg>Lys in Exon9 of SMAD4 gene
E: Transversion of T>G at codon 418; GCT>GCG; Ala>Ala in Exon9 of SMAD4 gene
Aga Syed Same
RESULTS-SMAD4

Sequence Results of SMAD4
Partial nucleotide sequence representing the mutant (Above) and normal (Below)
{Shown by asterik and arrows}, of Deletion of AGACA pentamer at codons 415/416
in Exon9 (MCR) of SMAD4 gene.
Aga Syed Same
RESULTS-SMAD4

Eleven out of sixteen (68.75%) of the SMAD4 gene mutants
were found to have mutations in KRAS gene as well.
There were nine (56.25%) tumors which had both KRAS
activating mutations as well as SMAD4 single point mutations.
Furthermore, in case of advanced/higher grade tumors (C+D =
48), KRAS gene mutations was found in 15 (31.25%) and
SMAD4 gene was found to be mutated in 14 (29.2%) tumors.
Also, 9 (18.75%) of C + D grade tumors had mutations in both
KRAS as well as SMAD4 gene as compared to only 2 (5.2%) of
A+B grade tumors.
The mutant status of KRAS and SMAD4 gene was found to be
significantly associated with the higher tumor grade (C+D) (P
value = 0.03).
Aga Syed Same
RESULTS-SMAD4Vs
KRAS

Correlation of SMAD4 with
KRAS
Correlation of SMAD4 gene status versus KRAS gene status.
SMAD4 Status
Wild
N = 70
Mutant
M= 16
OR; P Value; CI
(95%)
KRAS Status
Wild; n= 65
Mutant; n = 21
60 (92.3%)
10 (47.6%)
5 (7.7%)
11(52.4%)
0.07; 0.00003; 0.021-
0.26
Correlation of tumor grade versus SMAD4 gene status versus KRAS gene status.
Mutants
χ2, P valueKRAS
N=21
SMAD4
N=16
Both
N=11
Tumor Grade
A+B = 38
C+D = 48
6 (28.6%)
15 (71.4%)
2 (12.5%)
14 (87.5%)
6.63, 0.03 2 (18.2%)
9 (81.8%)
OR=0.28; 95%
CI=0.05-1.37
Aga Syed Same
RESULTS-SMAD4Vs
KRAS

Mutational spectrum of the signal transducing
molecules in CRC in Kashmiri population
Mutational spectrum of the signal transducing molecules in
CRC in Kashmiri population in comparison with the world
wide trend.
Gene Worldwide Trend* Kashmir
APC >60% 12.8%
β-catenin 20-40% 8.1%
KRAS >60% 24.4%
Braf 20% 0%
SMAD4 20% 18.6%
* Source: atlasgeneticsoncology.org
Percentage is for sporadic CRC
RESULTS
Aga Syed Same

Methylation Spectrum of Signal
RESULTS

MS PCR Analysis of APC 1A
promoter
Representative picture of PAGE analysis of APC 1A Promoter methylation.
Lane M: 50 bp molecular ladder (Prominent bands = 250bp & 500bp)
Lanes 1-14: Amplicons from paired samples
Case 1 & 5: Unmethylated 1A APC promoter
Case 2, 3, 4 & 6: Methylated 1A APC promoter
C1 & C2: Internal Unmethylated and Methylated Human DNA controls
RESULTS-APC

Representative picture of PAGE analysis of APC 1B Promoter methylation.
Lane M: 50 bp molecular ladder (Prominent bands = 250bp & 500bp)
Lanes 1-14: Amplicons from paired samples
Case 1, 5 & 7: Unmethylated 1B APC promoter
Case 2, 3, 4 & 6: Methylated 1B APC promoter
RESULTS-APC MS PCR Analysis of APC 1B
promoter

We found a significant association of APC
methylation status with the age group, tumor
location (colon) and tumor grade (C+D).
Clinico-epidemiological variables of the 86 colorectal carcinoma patients versus 47
hypermethylated penotypes of APC (1A&1B promoter) gene.
Variable Total
n = 86
MutantsΨ
n = 11 (12.79%)
Methylated#
n = 47 (54.65%)
P value*
Age group
≤60
>60
52 (60.5%)
34 (39.5%)
5
6
20
27
0.030
Tumorlocation
Colon
Rectum
36 (41.9%)
50 (58.1%)
8
3
29
18
0.030
Nodal status
Involved
Not Involved
48 (55.8%)
38 (44.2%)
6
5
38
9
0.004
Tumor grade
A+B
C+D
38 (44.2%)
48 (55.8%)
5
6
9
38
0.004
ΨOther than G>A transistion at codon 1492; #Either 1A or 1B promoter hypermethylation; *Fisher’s
two tailed test.
RESULTS-APC
Methylation

APC methylation status in 86 colorectal carcinoma cases in
Kashmiri population.
APC Promoter Status Cases n = 86
Either Methylated (1A or/& 1B)
Only 1A Methylated
Only 1B Methylated
Both Methylated
Neither Methylated
47 (54.65%)
9/47 (19.1%)
15/47 (31.9%)
23/47 (48.9%)
39 (45.35%)
Correlation of APC mutation status versus APC methylation status.
APC gene Status
OR; CI (95%); P ValueWild
W = 75
MutantΨ
M= 11
APC Promoter Methylation#
Unmethylated; n= 39
Methylated; n = 47
32 (42.7%)
43 (57.3%)
7 (63.6%)
4 (36.4%)
2.35; 0.63-8.72; 0.22
ΨOther than G>A transistion at codon 1492
#Either 1A or 1B promoter hypermethylation
RESULTS-APC
Methylation

Representative picture showing the hypermethylation status of the SMAD4 promoter.
Lane Ma: 50bp molecular marker (Prominent bands = 250bp & 500bp)
Lane Mb: 100bp molecular marker (Prominent band = 500bp)
Lane 1, 3, 5 & 7: Amplicons of the undigested template
Lane 2, 4, 6 & 8: HpaII treated gDNA PCR amplicons.
RESULTS–SMAD4
Methylation

Hypermethylation analysis of
SMAD4 gene carried out
using HpaII/MspI differential
restriction digestion did not
reveal any sort of methylation
in the CpG Islands of the
SMAD4 promoter
RESULTS–SMAD4
Methylation

Polymorphism of TP53
Codon 47 & 72
RESULTS

Representative gel picture of amplicons of
TP53 codon 47 target region (201/185bp).
Lane M: 50bp ladder (Two Bands = 250 & 500bp)
Lane 1-6: Amplicons from DNA’s of different
tumor tissues.
Restriction fragment length polymorphism analysis
of TP53 Pro47Ser SNP using MspI enzyme.
Lane M: 50bp ladder (Two Bands = 250 & 500bp)
Lane 1-6: Restriction digestion products; Wild
(Pro/Pro) is cleaved by Msp1 enzyme yielding two
fragments 156/140 and 45 bp while as mutant
(Ser/Ser) yields 201/185bp fragment.
Lane1 shows mutant (Ser/Ser) form, Lanes 2-6 shows
wild (Pro/Pro) form of SNP.
RESULTS–TP53
Polymorphism

Representative gel picture of amplicons of TP53
codon 72 target region (279bp).
Lane M: 100bp ladder (Prominent Band = 500bp)
Lane 1-6: Amplicons from DNA’s of different tumor
tissues.
Representative gel picture of restriction fragment
length polymorphism analysis of TP53 Arg72Pro
SNP using BstUI enzyme.
Lane M: 100bp ladder (Middle Band = 500bp)
Lane 1-6: Restriction digestion products; Wild
(Arg/Arg) is cleaved by BstUI enzyme yielding two
fragments 119, 160bp while as mutant ( Pro/Pro)
yields 279bp fragment.
Lanes 1 & 6 shows heterozygous (Arg/Pro) form,
Lanes 2 & 3 shows mutant form and Lanes 4 & 5
show wild (Arg/Arg) form of SNP.
RESULTS–TP53
Polymorphism

Genotype frequencies of TP53 gene polymorphisms in cases & controls
Cases
(n= 86)
Controls
(n=160)
P Value
TP53 Pro47Ser
C>T
Pro/Pro 81 (94.2%) 156 (97.5%) 0.166
Pro/Ser 0 0
Ser/Ser 5(5.8%) 4 (2.5%)
TP53 Arg72Pro
G>C
Arg/Arg 9 (10.5%) 65 (40.6%) 0.000001
Arg/Pro 37 (43.0%) 63 (39.4%)
Pro/Pro 40 (46.5%) 32 (20.0%)
Genotype frequencies of TP53 Codon 72 gene polymorphism in cases & controls and their
associations with the risk of CRC
TP53 Codon 72
Genotype
Cases
(n= 86)
Controls
(n=160)
OR(95% CI )
Arg/Arg 9 (10.5%) 65 (40.6%) 1.00
Arg/Pro 37 (43.0%) 63 (39.4%) 3.81 (1.75-8.32)
Pro/Pro 40 (46.5%) 32 (20.0%) 7.92 (3.51-17.87)
Arg/Pro + Pro/Pro 77 (89.5%) 95 (59.4%) 5.2 (2.5- 10.8)
RESULTS–TP53
Polymorphism

Partial nucleotide sequences
(Forward) of the TP53 Arg72Pro SNP.
•Homozygous CGC (Wild)
•Heterozygous CNC
•Homozygous CCC (Variant)
RESULTS–TP53
Polymorphism

Association between TP53 codon 72 phenotypes and clinic-pathologic characteristics
Variables
Cases (n=86)
n=86 Arg/Arg
9 (10.5%)
Arg/Pro
37 (43.0%)
Pro/Pro
40 (46.5%)
χ2 ; P Value
Tumor location
Colon
Rectum
36 (41.9%)
50 (58.1%)
3
6
27
10
6
34
26.84, 0.000001
Nodal status
Involved
Not Involved
48 (55.8%)
38 (44.2%)
4
5
11
26
33
7
22.23, 0.000015
Tumor grade
A+B
C+D
38 (44.2%)
48 (55.8%)
5
4
26
11
7
33
22.23, 0.000015
Bleeding
PR/Constipation
Yes
No
60 (69.8%)
26 (30.2%)
4
5
19
18
37
3
18.486, 0.0001
*One was SCC
RESULTS–TP53
Polymorphism

Wnt Pathway GenesDISCUSSION
APC gene to be aberrated in only 12.79% (11/86) colorectal
carcinomas, which was quite low than the reported frequency
by different researchers (Narayan & Roy, 2003; Beroud et al.,
1996; Rowan et al., 2000; Fearnhead et al., 2001;
Luchtenborg et al., 2004; Jeon et al., 2008; Smith et al.,
2002).
The low mutation frequency of APC gene suggests that
mutations may not be the foremost genetic aberration to be
implicated in the development of colorectal cancer in our
ethnic population.
We found a single nucleotide polymorphism (G>A) at codon
1492 in 72 (83.7%) CRC cases. This is the novel finding of our
study as it has not been reported from any part of the world.

We found a low frequency 8.1% (7 of 86) of β-catenin
mutations in CRC. These results were in tune with the
already present literature (Samowitz et al., 1999;
Miyaki et al., 1999; Johnson et al., 2005; Iwao et al.,
1998).
Exon 3 of β-catenin gene contains the regulatory
domain which is the hot spot for the genetic
aberrations.
Mutations in this exon have been reported in various
tumors resulting in its nuclear accumulation thereby
resulting in the progression of tumor (Narayan & Roy,
2003; Abraham et al., 2001; Munemitsu et al., 1995).

The mutations in hot spot codons – 32, 33, 41, 45, 49
in exon 3 of β-catenin gene results in an amino acid
change at GSK-3β phosphorylation sites which in turn
affects the phosphorylation resulting in the decreased
sequestration of β-catenin by APC (Munemitsu et al.,
1995).
Furthermore, the mutation affecting codon 45
(TCT>TTT; Ser>Phe) was present in lynch syndrome
patients as has been reported previously (Miyaki et
al., 1999; Johnson et al., 2005).
β-catenin and APC gene mutations were mutually
exclusive that further corroborated with the already
present literature (Narayan & Roy, 2003; Samowitz et
al., 1999; Iwao et al., 1998; Sparks et al., 1998; Morin
et al., 1997).

CpG island hypermethylation is one of the important
mechanisms of gene inactivation, cancer cell lines
have in general demonstrated an increased frequency
of hypermethylation compared with primary tumors
(Paz et al., 2003).
Both promoter hypermethylation and mutation are
responsible for the inactivation of tumor suppressor
genes in tumorigenesis (Esteller et al., 2000, 2002;
Karpiñski et al., 2008; Jones & Baylin, 2002).
A number of studies on colorectal cancer around the
globe have demonstrated the role of promoter
hypermethylation of number of different genes in
development and progression of colorectal carcinoma
(Lind et al., 2004; Lee et al., 2004; Toyota et al.,
1999).

Promoter hypermethylation of APC like other genes
plays a pivotal role in the inactivation of APC which in
turn enhances the tumor development (Chen et al.,
2005; Kang et al., 2003).
We found the hypermethylation in 54.65% (47 of 86)
CRC cases, this is consistent with other major studies
of the world, although markedly higher than reported
ones.
This may be due to the fact that the mutational
inactivation of APC gene is less in our population and
also because our population is exposed to a special
set of environmental challenges like extreme
temperature, high altitude, special food habits and
exposure to agricultural bye products like pesticides,
nitrosamines etc (Siddiqi et al., 1989).

MAP Kinase Pathway GenesDISCUSSION
Mutations in the KRAS oncogene are thought to occur
at an early stage in the adenoma-carcinoma
sequence, with the frequency of mutations increasing
with the adenoma size (Servomaa et al., 2000).
The frequency of mutations in the KRAS oncogene
has been reported to vary between 20 and 60%
(Vogelstein et al., 1988; Fearon & Vogelstein, 1990).
We found 24.4% mutation frequency of KRAS in our
population. 52.4% in codon 12, 23.8% in codon 13
and 9.5% each in codon 16 & codon 19. It is in tune
with few studies (Servomaa et al., 2000; Morin et al.,
1994).

The low frequency of KRAS mutations suggest that in
Kashmir, KRAS mutation may not be aggressively
involved in carcinogenesis and that the etiological
factors for CRC in Kashmir are likely to be different.
The substitutions of 12 and 13 amino-acid residues in
Ras altered its GTPase activity to a different extent
and/or its ability to interact with its regulators,
depending upon the substituted amino-acid residue
(Al-Mulla et al., 1998).
Replacement of Glycine 12 of Ras with any amino
acid, except proline, causes the biochemical activation
of Ras by the reduction of its intrinsic GTPase activity.

In addition to the commonly reported activating
mutation our analysis of KRAS gene also revealed a
novel transversion of A>T in codon 16 (Lys>Stop) and
an activating mutation in codon 19 (Leu to Phe).
This study is the first to report two novel mutations of
this nature. Similar kind of mutations have been
previously reported from this part of globe (Wang et
al., 2003), where codons 15, 18, 20 and 30 of KRAS
have been implicated in the tumorigenesis of CRC.
Wang et al in their study showed that codon 15 Kras
mutant protein has less GTPase activity than that of
the wild-type Kras protein due to a lack of response to
GAP induction owing to its less sensitivity to GAP.

TGF-β Pathway GenesDISCUSSION
The overall mutation rate of mutation cluster region
(MCR) of SMAD4 gene was found to be 18.6% (16 of
86), which is in tune with previous study (Miyaki et al.,
1999).
We found that 15 out of 16 mutations of SMAD4 gene
occurred in C-terminal region of SMAD4 gene which
codes for MH2 domain of Smad protein. These results
are in conformation with the previous studies (Miyaki
et al., 1999; Koyama et al., 1999; Christine et al.,
2004).
The MH2 domain of Smad4 protein is a multifunctional
region that mediates differential association with a
wide variety of proteins. Many of these interactions
serve to provide specificity and selectivity to Smad
function.

Another salient feature of the mutations found in this
study was the deletion of AGACA pentamer in exon 9
of the SMAD4 gene in one tumor tissue from a Familial
Adenomatous Polyposis case.
This affected codon is 415/16 of the SMAD4 gene
reflecting its effect on MH2 domain of Smad4 protein.
Similar type of deletion has also been reported
previously by Miyaki et al., 1999.
This tumor tissue was found to be KRAS mutant.

We also found that out of 16 SMAD4 mutants 11
(52.4%) were KRAS mutants also.
The cross talk mechanism between these two
pathways has been experimented upon and
conceptualized previously (Yue et al., 1999).
Cross-talk between RAS and TGF-β signaling has
been reported to play important roles in various
physiological and pathological processes, and RAS
signal has been reported to regulate TGF-β signaling
both positively and negatively (Pardali & Moustakas,
2007).
RAS transformation has been reported to confer
resistance to growth inhibition by TGF- β (Horiguchi et
al., 2009; Schwarz et al., 1998; Filmus et al., 1993).

We found a significant association between the
mutant statuses of the SMAD4 gene with that of the
KRAS gene, thereby suggesting that similar the cross
talk between the MAP Kinase and TGF-β pathways
might play a role in the progression of CRC to
advanced stage cumulatively in our population, (Yue
et al., 1999; Pardali & Moustakas, 2007; Javelaud et
al., 2005).
Janda et al., (2002 )reported that hyperactivation of
the Raf-MAP kinase pathway synergizes with TGF-β
signaling and induces epithelial-mesenchymal
transition (EMT) and accelerates the tumorigenesis
and metastasis of the otherwise normal cells.

TP53 PolymorphismDISCUSSION
Several studies have been carried out on the
association of TP53 polymorphism with the increased
risk for various cancers (Irarrazabal et al., 2003; Lee et
al., 2000; Hiyama et al., 2002; Zhu et al., 2007;
Sjalander et al., 1996; Thomas et al., 1999).
However in Colorectal Cancer the data available on
the TP53 polymorphic status is somewhat ambiguous,
where several studies found no association
(Hamajima et al., 2002), others reported an inverse
association (Perez et al., 2006), whereas other
indicated a positive association (Gemignani et al.,
2004).
this study, we assessed the two most common SNPs
of TP53 - Pro47Ser & Arg72Pro in an ethnic Kashmiri
population for the first time.

In case of TP53 Pro47Ser SNP, we found that Ser/Ser
frequency was 2.5% in controls whereas it was 5.8%
in cases, clearly showing a non-significant association
of this SNP with the colorectal cancer.
This observation was in contrast with the previous
study, where significant association was observed
between the mutant Ser47 phenotype with cancer risk
(Felley-Bosco et al., 1993).

In case of TP53 Arg72Pro , wild-type Arg/Arg
frequency was 40.6% in controls whereas it was
11.6% in cases, and mutant Pro-Pro frequency was
20.0% in control whereas it was 45.4% in cases, thus
Pro/Pro mutant phenotype shows a significant
association with the colorectal tumor development
(P=0.000001)
We also found that risk of developing CRC in case of
Pro/Pro genotype was 7.92 {95% CI = 3.51-17.87)
times that that of Arg/Arg genotype. The overall risk of
CRC development in case of Pro allele was 5.2 times
{95% CI = 2.5-10.8} than that of Arg allele.

Codon 72 polymorphism of TP53 is located within a
proline-rich region of the p53 protein and is functionally
important in the growth suppression and apoptosis
(Katkoori, 2009).
Furthermore it has been shown that Arg72 form of p53
has 15-fold enhanced capacity to induce apoptosis
than Pro72, which is due to more efficient localization
of the Arg72 form to mitochondria than the Pro72 form
(Thomas, 1999; Dumont, 2003; Murphy, 2006).
Moreover, several earlier studies have reported a
significant association between Pro/Pro mutant
phenotype and the risk of developing colorectal
cancer (Sjalander et al., 1995; Gemignani et al., 2004;
Zhu et al., 2008 & 2007; Katkoori et al., 2009)
therefore our findings were in tune with these studies.

Another remarkable fact that has arisen from this study
is that our population has significantly high of Pro/Pro
allele frequency than Arg/Arg.
This may be due to the fact that we reside at high
altitudes under high hypoxic condition and extremities
of environment, especially high exposure to UV-rays.
This observation is similar to the previously reported
studies (Katkoori et al., 2009; Bojesen &
Nordestgaard, 2008) where high Pro/Pro mutant
phenotype was found to be the race specific
molecular prognostic marker in colorectal cancer
especially in the populations challenged by the
environment.

Furthermore, in this study we also found a significant
association between the Pro/Pro mutant status and
tumor location, nodal status/higher tumor grade and
Bleeding PR/Constipation, but not with other
clinicopathologic variables .
These results were similar as reported by various
authors previously (Katkoori et al., 2009; Zhu et al.,
2007 & 2008; Lung et al., 2004; Sjalander et al., 1995;
Schneider et al., 2004).

Molecules Implicated in Colorectal Cancer Pathogenesis
In Kashmiri Population
Aga Syed Same
CONCLUSION
The epigenetic silencing of the APC gene showed high
frequency in our population, that signifies its putative role in the
initial tumorigenesis and also in the development and
progression towards the advanced stage.
The frequency of genetic alterations of KRAS is moderate in our
population, playing a role in the colorectal tumorigenesis.
However, the Braf was not found to be mutated in our population.
There was statistically significant association of SMAD4 gene
aberrations with KRAS mutant status suggesting the involvement
of at least two molecules in the advanced tumor grade in
colorectal cancers.
TP53 polymorphism in codon 72 predisposes our population to
cancer, because of the inherent preferential selection of Pro/Pro
mutant allele at higher frequency of 20% in our general
population.

Molecules Implicated in Colorectal Cancer Pathogenesis
In Kashmiri Population
Aga Syed Same
CONCLUSION
The multi-step model of CRC carcinogenesis holds well in explaining the
CRC tumorigenesis in our Kashmiri population but with a slight difference
in manner and frequency of genetic aberrations of key tumor suppressor
genes and oncogenes

PUBLICATIONS
A. Syed Sameer, Saniya Nissar, Syed Mudassar and Mushtaq A.
Siddiqi.
A. Syed Sameer and Mushtaq A. Siddiqi.
MTHFR C677T polymorphism and colorectal
cancer predisposition in Kashmiri population
Genetics & Molecular Research. 2011. In Press
SMAD4 gene promoter is not hypermethylated in
Colorectal Cancer of Kashmiri Population
Saudi J of Gastroenterology. 2011. In Press
Aga Syed Same

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