Introduction
There are numerous genetic pathways, which affect
colorectal cancer (CRC) initiation and progression. These include
the chromosomal instability pathways, which are associated with
accumulation of activating mutations in oncogenes, such as
KRAS, BRAF and PI3KCA or inactivation of tumor
suppressor genes, such as APC and p53 (1,2).
Furthermore, aberrant methylation of gene promoter regions has been
widely investigated and these epigenetic events within certain
signaling pathways result in gene suppression and contribute to the
development of CRC (3,4).
Genetic variation, such as single nucleotide
polymorphisms (SNPs), is hypothesized to contribute to individual
variations in CRC susceptibility (5). Polymorphic variants of genes are
significant factors that mediate inflammatory responses and
facilitate CRC progression (6). The
prognosis of CRC is dependent on the extent of local and metastatic
tumor spread and the degradation of the extracellular matrix (ECM)
that surrounds cancerous tissue. Matrix metalloproteinases (MMPs)
have fundamental roles in the degradation of basal membranes and
the ECM, as well as being associated with tumor invasion and a poor
prognosis (7). Our previous study
demonstrated that the gene polymorphism of the MMP12 gene is
associated with a higher risk of disseminated CRC (8).
Mitogen-activated protein kinase (MAPK) signaling
pathways (2,9) consist of a large family of
serine-threonine kinases, which respond to extracellular signals,
such as cellular stress and growth factors. These signals control
fundamental cellular processes, such as cell proliferation,
differentiation, cell survival and are considered to be significant
in the progression of different types of cancer (10), including CRC (2,9,10). p38
kinases are members of the MAPK family and consist of four isoforms
(α, β, γ and δ) (9–11). An increased expression level of
p38 has been observed in CRC (12) and breast cancer (13) patients.
Recently, it was shown that an SNP,
rs2235356, −1628A→G, in the promoter region of
the p38β gene was correlated with an increased risk of CRC
in a Chinese population (14). In
the present study, this SNP was analyzed to assess its value as a
risk factor and as a predictor of disease outcome in Swedish CRC
patients.
Patients and methods
Patients, controls and ethical
procedures
The present study collected blood samples from 389
consecutive patients from southeastern Sweden who underwent
surgical resection for primary colorectal adenocarcinomas at the
Department of Surgery, Ryhov County Hospital (Jönköping, Sweden)
between 1996 and 2013. Clinicopathological characteristics of the
patients were obtained from surgical and pathological records. The
investigation was approved by the local Ethical Committee of the
Faculty of Health Sciences (Dnr. 2013/271-31; Linköping, Sweden)
and informed consent was obtained from the participants.
Patient groups
The patients comprised 212 males and 177 females
with a mean age of 70 years (range, 25–93 years). The tumors were
located in the colon in 218 cases and in the rectum in 171 cases,
and were classified by Dukes’ classification system (15): Stage A, n=65; stage B, n=144; stage
C, n=121; and stage D, n=59. Blood donors (n=517) with no known
history of CRC, who were from the same geographical region as the
patients with CRC were selected as control subjects. The control
group consisted of 255 males and 262 females, with a mean age of 60
years (range, 33–79 years). Blood samples were centrifuged using
Hettich Universal 320R Centrifuge (Andreas Hettich GmbH & Co.
KG, Tuttligen, Germany) at 1500 × g for 10 min to separate the
plasma and blood cells and were stored at −78°C.
DNA extraction and genotype
determination
Genomic DNA was isolated from the blood samples
using the QIAamp DNA Blood kit (Qiagen, Valencia, CA, USA). DNA
samples were genotyped using the LightSNiP rs2235356 genotyping
assay (TIB Molbiol, Berlin, Germany). DNA (10 ng) was mixed with
Reagent Mix, LightCycler® FastStart DNA Master HybProbe
(Roche Diagnostics GmbH, Mannheim, Germany), 5.7 μl H2O
and 0.8 μl MgCl2 and amplified using the
LightCycler® 480 Real-Time PCR system (Roche Diagnostics
GmbH) according to the manufacturer’s instructions.
Statistical analysis
Differences in the frequencies of the p38β
gene polymorphism between patients and the control group, as well
as between clinical characteristics within the CRC subgroup were
analyzed using the χ2 test and the Hardy-Weinberg
equilibrium was assessed with regard to the genotypes. Survival
analysis was performed using Cox’s regression and Kaplan-Meier
analysis was conducted with the log-rank test. Statistical analysis
was performed using SPSS statistical software (version 19; IBM,
Armonk, NY, USA) and P<0.05 was considered to indicate a
statistically significant difference.
Results
Genotype distribution
The frequencies of the gene polymorphisms in the
patients and the control group subjects indicated no significant
difference in the genotype distribution or allelic frequencies
(Table I). The genotypes in the CRC
patients and the control group subjects were not associated with
clinical characteristics, such as age and gender (data not shown).
When subdividing the patients into groups of colon and rectal
cancer, or localized Dukes’ A and B and disseminated Dukes’ C and D
disease, no significant difference was identified between location
and disease with regard to the genotypes and alleles (Table II). In addition, analysis using the
Kaplan-Meier method demonstrated that the p38β genotypes
were not associated with survival rate (data not shown).
 | Table IGenotypic and allelic distribution of
the p38β gene polymorphism (−1628A→G) in
patients with CRC and in healthy control subjects. |
Table I
Genotypic and allelic distribution of
the p38β gene polymorphism (−1628A→G) in
patients with CRC and in healthy control subjects.
| A, Genotype
distribution |
|---|
|
|---|
| A→G | CRC, % (n=389) | Controls, %
(n=517) |
|---|
| A/A | 28.3 (110) | 24.9 (129) |
| A/G | 45.5 (177) | 51.3 (265) |
| G/G | 26.2 (102) | 23.8 (123) |
|
| B, Allele
distribution |
|
| Variant | CRC, % (n=778) | Controls, %
(n=1034) |
|
| A | 51.0 (397) | 50.6 (523) |
| G | 49.0 (381) | 49.4 (511) |
| Table IIGenotypic and allelic distributions of
the p38β gene polymorphism (−1628A→G)
regarding tumour location and disease stage in patients with
CRC. |
Table II
Genotypic and allelic distributions of
the p38β gene polymorphism (−1628A→G)
regarding tumour location and disease stage in patients with
CRC.
| A, Genotype |
|---|
|
|---|
| Location | Stage |
|---|
|
|
|
|---|
| A→G | Colon % (n) | Rectum % (n) | Dukes’ A + B %
(n) | Dukes’ C + D %
(n) |
|---|
| A/A | 28.4 (62) | 28.1 (48) | 28.2 (59) | 28.3 (51) |
| A/G | 45.0 (98) | 46.2 (79) | 46.0 (96) | 45.0 (81) |
| G/G | 26.6 (58) | 25.7 (44) | 25.8 (54) | 26.7 (48) |
| Total | (218) | (171) | (209) | (180) |
|
| B, Allele |
|
| Location | Stage |
|
|
|
| Variant | Colon % (n) | Rectum % (n) | Dukes’ A + B %
(n) | Dukes’ C + D %
(n) |
|
| A | 50.9 (222) | 51.2 (175) | 51.2 (214) | 50.8 (183) |
| G | 49.1 (214) | 48.8 (167) | 48.8 (204) | 49.1 (177) |
| Total | (436) | (342) | (418) | (360) |
Neither the patient nor the control group showed a
significant deviation in genotypic frequency as assessed by the
Hardy-Weinberg equilibrium (data not shown).
Discussion
The p38 MAPKs are involved in inflammation,
proliferation, differentiation, and cell death (2,9–11) and
increased levels have been observed in CRC patients (12). MMPs are fundamental in the
degradation of the ECM and are associated with tumor invasion and a
poor prognosis (7). The p38 MAPK
signaling pathways have been proposed to promote metastasis
(16) by activating different
transcription factors. One of these transcription factors,
activator protein-1 (AP1) (11,16)
appears to be important, as it controls MMP expression (16–19)
and may impact CRC progression via tumor invasion (20).
To the best of our knowledge, studies are limited
regarding the association between p38 genetic polymorphisms and the
risk of CRC. However, it was recently demonstrated that an SNP
(rs2235356, −1628A→G) in the promoter region
of the p38β gene was associated with an increased risk of
CRC in a Chinese population (14).
To elucidate whether this SNP is correlated with the risk of CRC in
other ethnic groups, this particular SNP was analyzed in Swedish
patients with CRC. In the present study, clinicopathological
variables, such as tumor localization, stage and survival were
evaluated, which is similar to a previous study by Huang et
al (14).
In the current study, it was identified that the
genotype distribution and allelic frequencies of patients were not
significantly associated with CRC when compared with the control
subjects. Functionally, the −1628A→G p38β gene
polymorphism may influence CRC progression via the p38 MAPK
signaling pathways as a regulator of the proliferation, survival
and metastasis. In addition, no significant correlation between the
genotypes and survival rate or disseminated disease was detected.
Furthermore, the genotypes were not identified to be associated
with other clinical parameters.
In conclusion, the present study indicated that the
−1628A→G polymorphism in the promoter region of the
p38β gene does not appear to be a useful tumor marker that
reflects the clinical outcome in Swedish patients with CRC. In the
future it would be of interest to evaluate this SNP in a large
cohort of various ethnicities.
Acknowledgements
The present study was supported by grants from
Futurum, the Academy for Healthcare, County Council (grant no.
105891; Jönköping, Sweden), the Foundation of Clinical Cancer
Research (grant no. 110426-1; Jönköping, Sweden) and the University
College of Health Sciences (Jönköping, Sweden).
References
|
1
|
Al-Sohaily S, Biankin A, Leong R,
Kohonen-Corish M and Warusavitarne J: Molecular pathways in
colorectal cancer. J Gastroenterol Hepatol. 27:1423–1431. 2012.
|
|
2
|
Markowitz SD and Bertagnolli MM: Molecular
origins of cancer: Molecular basis of colorectal cancer. N Engl J
Med. 361:2449–2460. 2009.
|
|
3
|
Kondo Y and Issa JP: Epigenetic changes in
colorectal cancer. Cancer Metastasis Rev. 23:29–39. 2004.
|
|
4
|
Naghibalhossaini F, Zamani M, Mokarram P,
Khalili I, Rasti M and Mostafavi-Pour Z: Epigenetic and genetic
analysis of WNT signaling pathway in sporadic colorectal cancer
patients from Iran. Mol Biol Rep. 39:6171–6178. 2012.
|
|
5
|
Mammano E, Belluco C, Bonafé M, Olivieri
F, Mugianesi E, Barbi C, Mishto M, Cosci M, Franceschi C, Lise M
and Nitti D: Association of p53 polymorphisms and colorectal
cancer: modulation of risk and progression. Eur J Surg Oncol.
35:415–419. 2009.
|
|
6
|
Theodoropoulos G, Papaconstantinou I,
Felekouras E, Niketeas N, Karakitsos P, Panoussopoulos D, Lazaris
ACh, Patsouris E, Bramis J and Gazouli M: Relation between common
polymorphisms in genes related to inflammatory response and
colorectal cancer. World J Gastroenterol. 12:5037–5043. 2006.
|
|
7
|
Egeblad M and Werb Z: New functions for
the matrix metalloproteinases in cancer progression. Nat Rev
Cancer. 2:161–174. 2002.
|
|
8
|
Van Nguyen S, Skarstedt M, Löfgren S, Zar
N, Andersson RE, Lindh M, Matussek A and Dimberg J: Gene
polymorphism of matrix metalloproteinase-12 and -13 and association
with colorectal cancer in Swedish patients. Anticancer Res.
33:3247–3250. 2013.
|
|
9
|
Fang JY and Richardson BC: The MAPK
signaling pathways and colorectal cancer. Lancet Oncol. 6:322–327.
2005.
|
|
10
|
Dhillon AS, Hagan S, Rath O and Kolch W:
MAP kinase signaling pathways in cancer. Oncogene. 26:3279–3290.
2007.
|
|
11
|
Zarubin T and Han J: Activation and
signaling of the p38 MAP kinase pathway. Cell Res. 15:11–18.
2005.
|
|
12
|
Pailas S, Boissière F, Bibeau F, Denouel
A, Mollevi C, Causse A, Denis V, Vezzio-Vié N, Marzi L, Cortijo C,
et al: Targeting the p38 MAPK pathway inhibits irinotecan
resistance in colon adenocarcinoma. Cancer Res. 71:1041–1049.
2011.
|
|
13
|
Chen L, Mayer JA, Krisko TI, Speers CW,
Wang T, Hilsenbeck SG and Brown PH: Inhibition of the p38 kinase
suppresses the proliferation of human ER-negative breast cancer
cells. Cancer Res. 69:8853–8861. 2009.
|
|
14
|
Huang Q, Chen D, Song S, Fu X, Wei Y, Lu
J, Wang L and Wang J: A genetic variation of the p38β promoter
region is correlated with an increased risk of sporadic colorectal
cancer. Oncol Lett. 6:3–8. 2013.
|
|
15
|
Wu JS: Rectal cancer staging. Clin Colon
Rectal Surg. 20:148–157. 2007.
|
|
16
|
del Barco Barrantes I and Nebreda AR:
Roles of p38 MAPKs in invasion and metastasis. Biochem Soc Trans.
40:79–84. 2012.
|
|
17
|
Jormsjö S, Ye S, Moritz J, Walter DH,
Dimmeler S, Zeiher AM, Henney A, Hamsten A and Eriksson P:
Allele-specific regulation of matrix metalloproteinase-12 gene
activity is associated with coronary artery luminal dimensions in
diabetic patients with manifest coronary artery disease. Circ Res.
86:998–1003. 2000.
|
|
18
|
Benbow U and Brinckerhoff CE: The AP-1
site and MMP gene regulation: what is all the fuss about? Matrix
Biol. 15:519–526. 1997.
|
|
19
|
Simon C, Simon M, Vucelic G, Hicks MJ,
Plinkert PK, Koitschev A and Zenner HP: The p38 SAPK pathway
regulates the expression of the MMP-9 collagenase via
AP-1-dependent promoter activation. Exp Cell Res. 271:344–345.
2001.
|
|
20
|
Ashida R, Tominaga K, Sasaki E, Watanabe
T, Fujiwana Y, Oshitani N, Higuchi K, Mitsuyama S, Iwao H and
Arakawa T: AP-1 and colorectal cancer. Inflammopharmacology.
13:113–125. 2005.
|
