International Journal of Genetics and Molecular Biology Vol. 2(6), pp. 101-111, June 2010     
Available online at http://www.academicjournals.org/IJGMB 
ISSN 2006-9863 © 2010 Academic Journals 
Review 
Colorectal cancer, TGF-β signaling and SMADs 
A. Syed Sameer
1, 2
, Safiya Abdullah
1
, Mujeeb Z. Banday
3
, Nidda Syeed
1
 and  
Mushtaq A. Siddiqi
1
* 
1
Department of Immunology and Molecular Medicine, Sher-I-Kashmir Institute of Medical Sciences, Soura, Srinagar, 
Kashmir, India 190011. 
2
Department of Clinical Biochemistry, Sher-I-Kashmir Institute of Medical Sciences, Soura, Srinagar, Kashmir,  
India 190011. 
3
Department of Biotechnology, University of Kashmir, Hazratbal, Srinagar, Kashmir, India 190011. 
Accepted 31 March, 2010 
Colorectal cancer (CRC) being the commonest cancer, is the major cause of mortality and morbidity 
worldwide.  TGF-β pathway is one of the important pathways that play a prominent role in cell 
proliferation, differentiation, migration and apoptosis. Smad dependent  TGF-  β signaling cascade is 
responsible for the regulation and expression of almost 500 odd genes, which in turn play important 
role in the proper development of intestinal mucosa. Here in this review we have discussed the overall
machinery of the  TGF-  β pathway and the advances in the mutational research on  SMAD4 gene in 
cancers with special look on our own research in CRC cases of Kashmiri population. 
Key words: Colorectal cancer, Kashmir, SMADs, mutations, PCR-SSCP. 
INTRODUCTION 
CRC (CRC), also called colon cancer or large bowel 
cancer includes cancerous growths in the colon, rectum 
and appendix. CRC is a commonly diagnosed cancer in
both men and women and is the third most common form 
of cancer and the second leading cause of cancer-related 
death in the western world. CRC causes 655,000 deaths 
worldwide per year, including about 16,000 in the UK, 
and about 50,000 in United States, where it is the second 
most common site (after lung) to cause cancer death
(World Health Organization, 2006; American Cancer 
Society, 2008). 
Two kinds of observations indicate a genetic contribution to CRC risk: a) increased incidence of CRC 
among persons with a family history of CRC; and b) 
families in which multiple family members are affected 
with CRC, the pattern indicates an autosomal dominant 
inheritance of cancer susceptibility (Burt et al., 1996; 
Lynch et al., 1996; Utsunomiya et al., 1990; Herrera, 
1990; Schoen, 2000). 
*Corresponding author. E-mail: vc.tmuk@gmail.com. Tel: +91 
9419767768. Fax: +91-194-2403470. 
About 75% of patients with CRC have sporadic dis-ease, 
with no apparent evidence of having inherited the 
disorder. The remaining 25% of patients have a family 
history of CRC that suggests a genetic contribution, common exposures among family members, or a combination 
of both. Genetic mutations have been identified as  the 
cause of inherited cancer risk in some colon cancer–
prone families; these mutations are estimated to account 
for only 5 - 6% of CRC cases overall. It is likely that other 
undiscovered major genes and background genetic 
factors contribute to the development of CRC, in conjunction with non-genetic risk factors (NCI, 2008). 
High incidence rates are found in western world 
populations, that is Western Europe, North America, and 
Australia. The lowest rates of CRC are found in the subSaharan Africa, South America and Asia, but are 
increasing in countries adopting western life-style and 
dietary habits (Vainio et al., 2003). 
Colorectal tumors present with a broad spectrum of 
neoplasms, ranging from benign growths to invasive 
cancer, and are predominantly epithelial-derived tumors 
(that is, adenomas or adenocarcinomas). Pathologists 
have classified the lesions into three groups: nonneoplastic polyps,  neoplastic  polyps  (adenomatous  polyps, 102          Int. J. Genet. Mol. Biol. 
adenomas), and cancers (O'brien et al., 2004; Zauber et 
al., 2002). 
More than 95% of CRCs are carcinomas and among 
them 95% are adenocarcinomas. While there is no direct 
proof that most CRCs arise from adenomas, 
adenocarcinomas are generally considered to arise from 
adenomas (Howe  et al.,  1998) based upon these two 
important observations: a) benign and malignant tissue 
occur within colorectal tumors; and b) when patients with 
adenomas were followed for 20 years, the risk of cancer 
at the site of the adenoma was 25%, a rate much higher 
than that expected in the normal population (O'Brien et 
al., 1990; Winawer et al., 2000). 
The etiology of CRC is multifactorial, and is likely to 
involve the actions of genes at multiple levels along the 
multistage carcinogenesis process. Examples of genes 
involved in pathogenesis of CRC include  p53,  p16,  p14, 
APC,  β-catenin,  E-cadherin,  transforming growth factor
(TGF)-β,  SMADs,  MLH1,  MSH2,  MSH6,  PMS2,  AXIN, 
STK11, PTEN, DCC and K-RAS (Sayar et al., 2007). 
REVIEW 
CRC is a common disease in both men and women. 
Because 5% of persons are predisposed to development 
of CRC, this disease is an important public health issue. 
CRC is the third most common cause of cancer-related 
death in the western world (Paula et al., 2002; Zoe et al., 
2004). 
Worldwide, CRC represents 9.4% of all incident cancers in men and 10.1% in women. CRC, however, is not 
equally common throughout the world. If the westernized 
countries (North America; those in northern, southern, 
and western Europe; Australasia; and New Zealand) are 
combined, CRC represents 12.6% of all incident cancers 
in westernized countries in men and 14.1% in women.
Elsewhere CRC represents 7.7% and 7.9% of all incident 
cases in men and women respectively (Boyle et al.,
2001). 
The lifetime risk of developing colon cancer in the
United States is about 7%. Certain factors increase a 
person's risk of developing the disease. The most important of these are the age, diet, obesity, diabetes  and 
smoking, personal cancer history, alcohol consumption, 
large intestinal polyps, family history of colon cancer, 
race and ethnic background, genetic or family predisposition. Colon cancer is usually observed in one of three 
specific patterns: sporadic, inherited, or familial. Sporadic 
disease, with no familial or inherited predisposition, 
accounts for approximately 70% of CRC in the population. Sporadic colon cancer is common in persons older 
than 50 years of age, probably as a result of dietary and 
environmental factors as well as normal aging. Fewer 
than 10% of patients have an inherited pre- disposition to 
colon cancer. The inherited syndromes  include  those  in  
which colonic polyps are a major manifestation of disease 
and those in which they are not. The polyposis syndromes are subdivided into familial adenomatous polyposis 
(FAP) and the hamartomatous polyposis syndromes. The 
nonpolyposis predominant syndromes include hereditary 
nonpolyposis CRC (HNPCC) (Lynch syndrome I) and the
cancer family syndrome (Lynch syndrome II). Although 
uncommon, these syndromes provide insight into the 
biology of all types of CRC. The third and least understood pattern of colon cancer development is known  as 
familial colon cancer. In affected families, colon  cancer 
develops too frequently to be considered sporadic colon 
cancer but not in a pattern consistent with an inherited 
syndrome. Up to 25% of all cases of colon cancer may 
fall into this category (Paula et al., 2002). 
Familial adenomatous polyposis patients inherit a 
mutated copy of the adenomatous polyposis gene (APC), 
whereas hereditary non-polyposis colon cancer is caused 
by inheritance of defective DNA mismatch repair genes 
(MLH1, MSH2,  PMS2 and  MSH6). Germ line mutations 
of the LKB1/STK11 gene have been shown to cause the 
Peutz–Jeghers syndrome (PJS), and mutation of SMAD4
or ALK3 underly juvenile polyposis (JPS). In contrast, the
MYH-associated polyposis (MAP) syndrome has autosomal recessive inheritance and results from bi-allelic 
mutations in the  MYH gene. Taken together, these 
syndromes account only for ~2 - 6% of CRC cases. The 
great majority of CRCs do not have a recognizable 
inherited cause, but a number of studies have suggested 
a role for genetic factors in predisposition to a substantial 
minority of colorectal tumors. The relatives of patients 
with ‘sporadic’ CRC are themselves at increased risk of 
the disease and segregation analysis has suggested 
dominant inheritance of the uncharacterized susceptibility 
genes. (Cannon-Albright  et al.,  1988; Houlston et al.,
1992; Johns et al., 2001; Hans et al., 1996). 
CRC – An overview 
The majority of CRCs develop from benign pre-neoplastic 
lesions: the adenomatous polyps or adenomas. Progression from a benign adenoma to a malignant carcinoma
passes through a series of well-defined histological 
stages, which is referred to as the adenoma-carcinoma 
sequence (Vogelstein et al., 1988). Two major 
mechanisms of genomic instability have been identified 
that give rise to colorectal carcinoma development  and 
progression: chromosomal instability (CIN) and microsatellite instability (MIN). CIN is associated with a series 
of genetic changes that involve the activation of 
oncogenes as k-ras and inactivation of tumor suppressor 
genes as p53,  DCC/SMAD4 and  APC and contributes 
predominantly to carcinogenesis in the distal segments of 
the colorectum (conlin et al., 2005; Esteller et al., 2001; 
Hsieh  et  al.,  2005).  Familial   Adenomatous   Polyposis    Sameer et al.      103 
Table 1. Association of various aberrant molecules of TGF-β pathway with different cancers. 
TGF-β component  Cancers 
TGF-β Aggressiveness because of enhanced invasion and metastasis 
Type I receptor 
Implicated in all cancers; Colorectal (30%), gastric (15%), prostate, breast, lung, pancreatic, head 
and neck etc 
Type II receptor  Breast (16%), pancreatic, biliary, cervical 
Smad 2  Colorectal (11%), lung (7%), liver 
Smad4 
Implicated in all cancers; Pancreatic (50%), Colorectal (30%), Lung (10%), Breast, Ovarian, Gastric, 
Prostate, Esophageal, Liver, Head and Neck etc 
represents the hereditary syndrome dealing with  APC 
mutation (Vogelstein et al., 1988; Fearon et al., 1990). 
Mutations in DNA mismatch repair (MMR) genes result in 
a failure to repair errors that occur during DNA replication 
in repetitive sequences (microsatellites), resulting in an 
accumulation of frameshift mutations in genes that 
contain microsatellites. This failure leads to MIN  type of 
tumor and is the hallmark of hereditary non-polyposis 
CRC (HNPCC) (Boland et al., 1998). MIN is also found in 
12 - 15% of sporadic CRCs. In addition to the genetic 
disparity of CIN and MIN, MIN tumors are more 
frequently right-sided and poorly differentiated, and more 
often display unusual histological type (mucinous), and 
marked peri-tumoral and intra-tumoral lymphocytic 
infiltration (Dolcetti et al., 1999; Benatti et al., 2005). 
Classification and grading 
The most common colon cancer cell type is adenocarcinoma which accounts for 95% of cases. Other, rarer 
types include lymphoma and squamous cell carcinoma.
Cancers on the right side (ascending colon and cecum) 
tend to be exophytic, that is, the tumor grows outwards 
from one location in the bowel wall. Left-sided tumors 
tend to be circumferential, and can obstruct the bowel 
much like a napkin ring. Pathology has an essential role 
in the staging of CRC. Two classification systems are 
being used for the staging of the CRC- Dukes 
classification and TNM (Tumors/Nodes/Metastases) 
system. Dukes' classification, first proposed by Dukes et 
al., (1932), identifies the stages as: A - Tumor confined to 
the intestinal wall; B - Tumor invading through the
intestinal wall; C - With lymph node(s) involvement and D 
- With distant metastasis, which is the commonest in use 
still. There has been a gradual move from using Dukes’s 
classification to using the TNM classification system as 
this is thought to lead to a more accurate, independent 
description of the primary tumors and its spread (Hardy et 
al., 2001).  
GENETIC BACKGROUND OF CRC 
TGF-β signaling 
The TGF-β superfamily consists of more than 35 members in vertebrates, including TGF-β, BMPs (bone 
morphogenetic proteins), GDFs (growth differentiation 
factors), activins, inhibins, MIS (Mullerian inhibiting 
substance), nodal, and leftys (Table 1). These proteins 
were identified mainly through their roles in development; 
they regulate the establishment of the body plan and 
tissue differentiation through their effects on cell 
proliferation, differentiation and migration (Figure 1 and 
2). The growth inhibitory effect of TGF-β signaling in 
epithelial cells explains its role as a tumor suppressor in 
carcinomas, although TGF-β expression by tumor cells 
contributes to cancer progression as well (Derynck et al.,
2003; Massague et al., 1998). The TGF-β family ligands 
are translated as prepropeptide precursors with an  Nterminal signal peptide followed by the prodomain and 
the mature domain (Hogan et al., 1996; Padgett et al.,
1997; Change et al., 2002). 
TGF-β superfamily ligands signal through a family of 
transmembrane serine/threonine kinases known as the
receptors for the TGF-β superfamily, which are divided 
into two subfamilies: type I and type II receptors. The 
extracellular regions of these receptors contain about 150 
amino acids with 10 or more cysteines that determine the 
folding of this region (Chang et al., 2002; Wrana et al.,
1994; Lastres et al., 1996; Cheifetz et al., 1991; Segarini 
et al., 1989). 
TGF-β signaling pathways are broadly categorized into 
two types depending upon the main mediators in 
intracellular environment as Smad dependent and Smad 
independent.  Smad dependent pathway is the most 
characterized of all the TGF-β signaling pathways. In 
Smad dependent pathway signaling is initiated when the 
ligand in dimeric form induces assembly of a heteromeric 
complex of type II and type I receptors. The type II kinase 
then phosphorylates the type I   receptor  in  a  conserved 104          Int. J. Genet. Mol. Biol. 
Figure 1. Overview of TGF-β pathway (Courtesy: Biocarta.org). 
Figure 2.  Signaling specificity of TGF-B superfamily (Adapted from 
Moustakas A. et al. J. Cell Sci. 2001 114: 4359-4369). 
glycine–serine-rich domain (GS domain). This activates 
the type I kinase, which subsequently recognizes and 
phosphorylates members of the intracellular Smad signal 
transduction pathway. Activation of the Smad pathway 
occurs when the activated type I kinase associates  with 
the MH2 domain of specific R-Smads. The  type  I  kinase  then phosphorylates the R-Smads on a conserved 
carboxy-terminal SSXS motif. This causes dissociation of 
the R-Smad from the receptor, stimulates the assembly 
of a heteromeric complex between the phosphorylated RSmad and the Co-Smad, Smad4, and induces the 
nuclear accumulation of this heteromeric Smad complex. 
In the nucleus, Smads function to regulate transcriptional 
responses by directly interacting with a host of resident 
DNA binding proteins. Here, the R-Smads mediate the
interaction of the Smad complex with DNA binding 
proteins. Once recruited to specific regulatory elements, 
Smads can then stabilize ternary DNA binding complexes 
by contacting DNA at adjacent sites and can directly 
regulate transcription by recruiting coactivators or 
corepressors to the promoter. Thus, Smads function  to 
transmit signals directly from the cell-surface receptors 
into the nucleus, where they act as effectors of the 
transcriptional response to TGFβ-related factors (Grady 
et al., 2000; Attisano et al., 2000). 
SMAD4 – (Mothers against decapentaplegic homolog 
4 (drosophila)) and CRC 
SMAD4  gene - also known as  MADH4,  DPC4 &  JIP, is 
located on the long arm (q) of chromosome 18 at band 
21.1. The gene encompasses 49.5 kb of DNA with 13 
exons, out of which first two exons do not code for any 
amino acid and hence constitute 5’-UTR of the SMAD4 
gene.  SMAD4  mRNA transcript constitutes 3220 
nucleotides. The protein of  SMAD4 gene - Smad4
belongs to the Darfwin family of proteins which harbours 
two conserved amino- and carboxyl-terminal domains 
known as MH1 and MH2, respectively. Smad4 in the 
basal state is found mostly as a homo-oligomer, most 
likely a trimer. It is ubiquitously expressed within the 
human body. Smad4 is an intracellular mediator of TGF-β
family and activin type 1 receptor. Smad4 mediate TGF-β
signaling to regulate cell growth and differentiation. TGF-
β stimulation leads to phosphorylation and activation of 
Smad2 and Smad3, which form complexes with Smad4 
that accumulate in the nucleus and regulate transcription 
of target genes. By interacting with DNA-binding proteins, 
Smad complexes then positively or negatively regulate 
the transcription of target genes (Attisano et al., 2000, 
2001; Massague et al., 2000; Wrana et al., 2000; Shi, 
2001; Saffroy et al., 2004). 
The discovery of human homologues of the Drosophila
Mad gene, called Smad genes (Hahn  et al.,  1996), has 
been a milestone for understanding the genetics of  the 
CRC whether of familial origin or sporadic. It has opened 
the Pandora’s Box for both developmental and cancer
biologists. Mutations in two Smad family member genes – 
Smad4 (also known as  DPC4) and Smad2 (also known 
MADR2, and hMAD-2) have been identified in human 
cancers   and  more  importantly  with  high  frequency  in  
Sameer et al.      105 
pancreatic and CRCs (Riggins et al., 1996). This raises 
the possibility that one or more of these genes can act as 
tumor suppressors as well as developmental regulators. 
Approximately 50% of pancreatic carcinomas, 20% of 
colon carcinomas, and 10% of lung cancers exhibit 
mutations in Smad4, and mutations in Smad2 have been 
found in ~7% of colorectal and lung cancers (Hahn et al., 
1996; Riggins et al., 1996, 1997; Uchida et al., 1996). 
The Smads are a group of related intracellular proteins 
which play a critical role in transmitting the signals from 
the transforming growth factor-β (TGF-β) superfamily 
located at the cell surface onto the nucleus (Attisano et 
al., 2000; Massague et al., 2000; Wrana et al., 2000; 
Dijke et al., 2000; Padgett et al., 1999; Zhang et  al., 
1999; de Caestecker et al., 2000). Although related to 
each other, Smads are structurally distinct from other 
intracellular effector proteins. The prototypic members of 
the Smad family,  Mad and  Sma, were first described in 
Drosophila and  Caenorhabditis elegans, respectively 
(Padgett et al., 1999). Related proteins in  Xenopus, 
humans, mice and rats were subsequently identified, and 
all family members are now known as Smads, a contraction of the invertebrate gene names. More recently, 
related proteins have also been described in zebra-fish 
and the helminth parasite Schistosoma mansoni (Raftery 
et al., 1999). 
There are eight Smad family members in mammals, 
and a search of human genome database suggests that
this represents the full complement. The eight human 
Smad genes have been mapped to four chromosomes. 
Three of the Smad genes - Smad2, Smad4 and Smad7 - 
are closely clustered at 18q21.1, a region that is 
frequently deleted in human cancers. Three are found on 
chromosome 15, with Smad3 and Smad6 mapping to 
15q21 - 22 and Smad5 to 15q31. The remaining Smad 
genes, Smad1 and Smad8, are located on chromosomes 
4 and 13, respectively (Attisano et al., 2001). Smads are 
ubiquitously expressed throughout development and in all 
adult tissues, and many of them (Smad2, Smad4, 
Smad5, Smad6 and Smad8) are produced from alternatively spliced mRNAs (Luukko et al., 2001). 
Functional studies have demonstrated that Smads, 
which range from about 400 to 500 amino acids in length, 
can be grouped into three subfamilies: a) the receptorregulated Smads (R-Smads: Smad1, Smad2, Smad3, 
Smad5, Smad8), which become phosphorylated by the 
type I receptors; b) the common Smads (co-Smads: 
Smad4), which oligomerise with activated R-Smads; and 
c) the inhibitory Smads (I-Smads: Smad6 and Smad7), 
which are induced by TGF-b family members. Each of 
these Smads plays a distinct role in the pathway (Figure 
3) (Attisano et al., 2001; Moustakas et al., 2001).
Smads  have  two  conserved domains, the  N-terminal 
Mad homology 1 (MH1) and C-terminal Mad homology 2 
(MH2) domains. The MH1 domain is highly conserved 
among R-Smads and Co-Smads; however, the N-terminal  106          Int. J. Genet. Mol. Biol. 
Figure 3. 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) 
(Adapted from Moustakas A. et al., J. Cell Sci. 2001 114: 4359-4369).
parts of I-Smads have only weak sequence similarity to 
MH1 domains. Sequence and structural analyses indicate 
that the MH1 domain is homologous to the diverse HisMe (histidine-metal-ion) finger family of endonucleases, 
and it may have evolved from an ancient enzymatic 
domain that had lost its catalytic activity but retained its 
DNA-binding properties (Grishin  et al.,  2001). The MH1 
domain regulates nuclear import and transcription by 
binding to DNA and interacting with nuclear proteins. The 
MH2 domain is highly conserved among all Smads. Its
structure contains several  α-helices and loops, which 
surround a  β-sandwich, and it resembles the fork head 
associated (FHA) domain, a phosphopeptide-binding 
domain common in transcription and signaling factors. 
The MH2 domain regulates Smad oligomerisation, recognition by type I receptors and interacts with cytoplasmic 
adaptors and several transcription factors (Shi Y,  2001; 
Moustakas et al., 2001; Li et al., 2000). 
SMAD4 and aberrations 
The role of Smad4 gene as an important tumor suppressor gene came out by the novel study of the allelotype loss in pancreatic adenocarcinoma (Shi Y, 2001). 
This study showed that about 90% of these tumors show 
allelic loss of chromosome 18q. In the same year another 
study identified the genetic target of these allelic losses 
as the DPC4 gene (DPC-Deleted in Pancreatic Carcinoma, locus 4). The study analyzed 338 tumors, originating 
from 12 distinct anatomic sites, for alterations in the 
DPC4  gene. An alteration of the  DPC4 gene sequence 
was identified in one of eight breast carcinomas and one 
of eight ovarian carcinomas.  DPC4 was found to be 
homozygously deleted in about 30% of pancreatic 
carcinomas and inactivated by intragenic mutation in 
another 20% of the tumors. The tissue restriction of 
alterations in  DPC4, as in many other tumor-suppressor 
genes, emphasizes the complexity of rate-limiting checkpoints in human tumorigenesis (Schutte et al., 1996). 
Smad4 was proposed to be a tumor suppressor gene 
that may function to disrupt TGF-β signaling. Mutant 
Smad4 proteins, identified in human carcinomas, were 
found to be impaired in their ability to regulate gene 
transcription. Most of Smad4 gene mutations in human 
cancer are missense, nonsense, and frameshift mutations at the mad homology 2 region (MH2) which interfere 
with the homo-oligomer formation of Smad4 protein and 
hetero-oligomer formation between Smad4 and Smad2 
proteins, resulting in disruption of TGF-β signaling (Table 
2) (Shi, 2001;  Woodford-Richens et al., 2001; Roth et al.,
2003). 
Moskaluk et al. (1997) later on described the optimized 
primers and conditions used in polymerase chain reaction  Sameer et al.      107 
Table 2. Nature of SMAD4 MCR region mutations in colorectal carcinoma patients (Reported from across the globe). 
SMAD4 Exon  Mutation  Amino acid change  Affected codon  Effect 
1  GGA>GTA  Gly>Val  64  MS 
1  GCT >GTT   Ala >Val  86  MS 
2  TAT>AAT  Tyr> Asn  94  MS 
2  TGG>CGG  Trp>Arg  99  MS 
2  AAA>AAAA  Insertion (1bp)  106  FS 
2  TGT>CGT  Cys>Arg  115  MS 
2  GCG>GAG  Ala>Glu  118  MS 
2  GCG>GTG  Ala>Val  118  MS 
2  TTA >TA  Deletion (1bp)  121  FS 
2  TTA >TTAA  Insertion (1bp)  121  FS 
2  GTC> GCC   Val to Ala  127  MS 
2  AAT>AAG  Asn>Lys  129  MS 
2  CGA>TGA  Arg>Stop  135  NS 
4  GGA>TGA  Gly>Stop  168  NS 
4  TAC>TAA  Tyr>Stop  195  NS 
4  CAG>TAG  Gln>Stop  245  NS 
4  ACT to A  Deletion (2bp)  259  FS 
6  ACT>ACTT  Truncation at codon 271/72  269-270  FS 
7  TGG>CGG  Trp>Arg  302  MS 
8  AGT>AAT  Aberrant splicing  Intron-Exon region  Splice site change 
8  TAC> TAA   Tyr >Stop  328  NS 
8  GAA>GCA  Glu>Ala  330  MS 
8  GAA>AAA  Glu>Lys  330  MS 
8  GAT>GGT  Asp>Gly  332  MS 
8  AAG>GAG  Lys>Glu  340  MS 
8  AT….AA>AA  Deletion (15bp)  339-343  FS 
8  GAGAGA>GAGA  Truncation at codon 339-40  336-338  FS 
8  GTT>GAT  Val>Asp  350  MS 
8  GAT>CAT  Asp>His  351  MS 
8  TAC>TGC  Tyr>Cys  353  MS 
8  GAC>GAA  Asp>Glu  355  MS 
8  CGC>AGC  Arg>Ser  361  MS 
8  CGC>CAC  Arg>His  361  MS 
8  TGT>AGT  Cys>Ser  363  MS 
8  GTT>GAT  Val>Asp  370  MS 
9  TGC>CGC  Cys>Arg  401  MS 
9  TTT>TCT  Phe>Ser  408  MS 
9  CAG>CACAG  Insertion (2bp)  410  FS 
9  AGACAGAG>AGAG  Deletion  415-16  FS 
9  GCA>GTA  Ala>Val  433  MS 
10  CAG>TAG  Gln>Stop  442  NS 
10  CGA>TGA  Arg>Stop  445  NS 
10  GC…..AGC>GC  Deletion (25bp)  447-455  FS 
10  CA…CT>CT  Deletion (28bp)  450-459  FS 
11  GGT>GTT  Gly>Val  491  MS 
11  GTT>TTT  Val>Phe  492  MS 
11  GAT>GCT  Asp>Val  493  MS 
11  CGC>CAC  Arg>His  497  MS 
11  TGC>TAC  Cys>Tyr  499  MS 108          Int. J. Genet. Mol. Biol. 
Table 2. Contd. 
SMAD4 Exon  Mutation  Amino acid change  Affected codon  Effect 
11  AAA>CAA  Lys>Gln  507  MS 
11  AGA>GGA  Arg>Gly  515  MS 
11  CTC>GTC  Leu>Val  533  MS 
11  GAT>TAT  Asp>Tyr  537  MS 
11  CTA>CGA  Leu>Arg  540  MS 
11  CT…CC>CC  Deletion (7bp)  540-542  FS 
MS: Missense mutation; NS: Nonsense mutation and FS: Frameshift mutation. 
and cycle sequencing of the entire DPC4/SMAD4 coding 
sequence. In another study, a subset of juvenile polyposis syndrome (JPS) families was identified to carry germ 
line mutations in  SMAD4 gene. The mutant  SMAD4 
proteins were predicted to be truncated at the carboxylterminus and lack sequences required for normal 
function. These results confirmed an important role for 
SMAD4 in the development of gastrointestinal tumors 
(Howe et al., 1998). However another study carried out in 
England (Houlston et al., 1998), having the same design 
as the previous one reported somatic missense 
mutations affecting codon 361 (CGC/arg→TGC/Cys) in 
DPC4/SMAD4 gene in juvenile polyposis tumors. 
In the same year, a study on the mutational spectrum 
of the  SMAD2 gene was carried out in National Cancer 
Center Institute, Japan on human colon cancers 
(Takenoshita et al., 1998). The study revealed that
though there was no mutation within all exons of the 
SMAD2  gene, two of 60 sporadic CRCs displayed 
deletions in the polypyrimidine tract preceding exon4. 
Deletions of this region were also detected in colon 
cancer cell lines, and were clustered within cells 
exhibiting microsatellite instability. 
Koyama et al. (1999) investigated the potential role of 
DPC4/SMAD4 gene in CRCs. LOH was identified in 50 - 
78% of the tumors that were informative for polymorphic 
markers in the region. Somatic mutations were identified 
in seven of those tumors: two frameshift mutations, a 1-
bp deletion (326 del T) in exon8 and a 1-bp insertion (50 - 
51 ins A) in exon1; two nonsense mutations, Arg445Ter 
in exon10 and Glu538Ter in exon11; and three missense 
mutations, Asn129Lys in exon 2, Tyr95Asn in exon 2,
and Asp355Glu in exon8. Three of the seven mutations 
were observed in the MH1 domain encoded by exons1 
and 2. The results demonstrated that inactivation of both 
alleles of the DPC4/SMAD4 gene occurs in a substantial 
proportion of advanced CRCs, and that the  DPC4/ 
SMAD4 gene probably exerts a tumor-suppressor effect 
for colorectal carcinogenesis that fulfills the criterion of 
the two-hit concept proposed by Knudson A.G. (1985). 
With time, a large number of researchers’ detected 
mutations in  SMAD2 and SMAD4 genes in some colorectal carcinomas (Riggins et al., 1997; Houlston et al.,
1992; Thiagalingam et al., 1996; Takagi et al., 1996; Mac 
Grogan et al., 1997), however the frequencies of these 
mutations have been found to be low, and the role of 
these genes in colorectal carcinogenesis is still unclear. 
In order to clarify the contribution of SMAD genes in 
colorectal carcinogenesis, Miyaki et al. (1999) analyzed 
mutations of Smad2, 3, 4, 6 and 7 in different stages of 
tumor. Their study revealed twenty-one Smad4 mutations 
and one Smad2 mutation, whereas mutation of Smad 3,
6 and 7 genes was not detected. Smad4 mutations 
included seven frameshift, one inframe deletion, four 
nonsense and nine missense mutations, 95% of which 
resulted in alteration of Smad4 protein regions included in 
homo-oligomer and hetero-oligomer formation. Frequencies of tumors with Smad4 mutation were 0/40 (0%) in 
adenoma, 4/39 (10%) in intramucosal carcinoma, 3/44
(7%) in primary invasive carcinoma without distant 
metastasis, 6/17 (35%) in primary invasive carcinoma 
with distant metastasis, and 11/36 (31%) in distant metastasis. In a similar study Yakicier et al. (1999) analyzed 
mutations in  SMAD2 and  SMAD4 in hepatocellular 
carcinoma (HCC). The study was carried out on 35 HCC 
and non-tumor liver tissues. The results revealed that 
three tumors displayed somatic missense mutations,  all 
of which were transistions of A: T →G: C type; two were 
in  SMAD4 (Asp332Gly and Cys401Arg) and one was in 
SMAD2 (Gln407Arg). 
By the start of the 21
st
 century the mutational analysis 
of  smad genes got a boost and large number of 
researchers reported the mutations in  SMAD2 and or 
SMAD4 genes in CRCs of sporadic or of familial type and 
of different grades (Woodford-Richens et al., 2001; Roth 
et al., 2003; Ohtaki et al., 2001; Howe et al., 2001, 2002, 
2004; Sayed et al., 2002). 
Christine et al. (2004) recently reported the mutational 
hot spot in SMAD4 gene and its functional consequences 
in human tumors. Their study concluded that the 
homozygous deletion, followed by inactivating nonsense 
or frameshift mutations, is the predominant form of
SMAD4 inactivation in pancreatic cancers. Among the 
naturally occurring SMAD4 missense mutations, the MH2 
domain is the most frequent target (77%) of missense 
mutations in human tumors. A mutational hot spot resides  Sameer et al.      109 
Table 3. Nature of SMAD4 MCR region mutations in colorectal carcinoma patients from Kashmir valley. 
SMAD4 Exon  Mutation  Amino acid change  Affected codon  Effect 
2  TGT>CGT  Cys>Arg  115  MS 
8  CGC>AGC  Arg>Ser  361  MS 
8  CGC>CAC  Arg>His  361  MS 
8  TTT>TTG Phe>Leu  362  MS 
8  TGT>AGT  Cys>Ser  363  MS 
8  GGTT>GAGTT  Insertion  341  FS 
8  CGC>CAC  Arg>His  361  MS 
9  TGG>GGG  Trp>Gly   419  MS 
9  AGACAGAG>AGAG  Deletion  415/16  FS 
9  AGA>AAA  Arg>Lys  415  MS 
10  CAG>TAG  Gln>Stop  442  NS 
10  GCT>GCC Ala >Ala  456  S 
10  CGA>TGA  Arg>Stop  445  NS 
11  AAAGGC>AATTGC  Lys> Asn; Gly>Cys  507/8  MS 
11  GGC>AGC  Gly>Ser  508  MS 
11  AAA>CAA  Lys>Gln  507  MS 
within the MH2 domain corresponding to codons 330 -
370, termed the mutation cluster region (MCR). These 
findings have important implications for in vitro functional 
studies, suggesting that the majority of missense 
mutations inactivate Madh4 by protein degradation in 
contrast to those that occur within the MCR (Christine et 
al., 2004). 
More recently a number of articles have been published 
on the SMAD4 mutations and their increased association 
with the CRC. One of the important observations has
been the identification of SMAD4 gene as the prognostic 
marker of the subtype of CRC. A recent article published 
in cancer letters by Qiu et al. (2007) identified the novel 
nonsense mutations in  SMAD4  gene located in exon 5 
codon 245 CAG (glut)  →TAG (stop) and in  SMAD2 in 
exon 8 at codon 276 TCG (ser) → TTG (leu). 
In our own study carried out on 86 primary colorectal 
carcinomas from Kashmiri population, we have found 16 
(18.6%) tumors harboring  SMAD4  mutations (Table 3). 
There were eleven missense mutations, one silent 
mutation, two nonsense mutations, one silent mutation 
and two frameshift mutations including eight transitions 
and four transversions. Among the two frame shift 
mutations, one was observed in codon 341 (exon 8) due 
to insertion of A and the other one in codons 415/416 
(exon 9) due to deletion of AGACA pentamer respectively 
(Table 2, Figure 1). Among the transistions, G:A>A:G 
substitutions were most prevalent followed by C:T>T:C. 
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 occured in exon 10 of SMAD4 gene. (Manuscript 
Submitted in BMC Cancer). 
In conclusion, considering the important role of SMAD4
in the colorectal carcinogenesis one can say that SMAD4
plays a role of important molecular gladiator in the 
development of normal mucosa. If however, this gladiator 
losses to the mutations then it creates havoc in the tissue 
morphology leading to the uncontrolled development  of 
mucosa which in turn leads to progression to tumor. As 
proved by most studies  SMAD4 may serve as the 
important prognostic molecule in CRC, laboratories 
across the world may use it for better treatment of the 
secondary CRC especially in identification and treatment 
of Dukes C patients and thus help in increasing the
overall survival of the patient.  
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