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ORIGINAL ARTICLE
Year : 2014  |  Volume : 10  |  Issue : 3  |  Page : 631-635

An evaluation of nuclear factor kappa B expression in colorectal carcinoma: An analysis of 50 cases


Department of Pathology, Armed Forces Medical College, Pune, Maharashtra, India

Date of Web Publication14-Oct-2014

Correspondence Address:
Nikhil Moorchung
Department of Pathology, Armed Forces Medical College, Sholapur Road, Pune - 411 040, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.139159

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 > Abstract 

Context: There is a rising trend in the incidence of (colorectal carcinoma) colorectal cancer (CRC) in India. Nuclear factor kappa B (NFkB) is a transcription factor which belongs to the Rel family. It has an impact on phenomena such as apoptosis, tumor progression and differentiation.
Aims: (1) To evaluate the grade and stage in 50 cases of colorectal carcinoma. (2) To evaluate the NFkB translocation into the nucleus of the cells. (3) To compare the benign and malignant areas with the degree of NFkB translocation and compare the translocation with the grade and pathological stage.
Subjects and Methods: The grade and stage of the tumors was evaluated. NFkB staining was performed on the tissues. The results of the immunostaining were analyzed semi quantitatively as a percentage of positive cells.
Statistical Analysis Used:  Statistical Package for Social Sciences 13.0 statistical package program (SPSS, Lead Technologies Inc, USA) for windows was used. Correlation was carried out using the Pearson's correlation co-efficient and the Chi-square test.
Results: There were 29 males (58%) and 21 females (42%). Most of the cases were well-differentiated adenocarcinomas (58%). There was a significant difference between NFkB translocation in the epithelial cells and lymphocytes in the benign and malignant areas (P - 0.04 and P - 0.001 respectively). There was a significant correlation between the grade of NFkB staining in the malignant epithelial cells with the tumor and nodal status (P - 0.001 and P - 0.001).
Conclusions: It is likely that NFkB is an important factor in the pathogenesis of CRC. Further studies including therapeutic intervention using strategies which prevent activation of NFkB in colorectal carcinoma patients will tell if we could alter the course of the disease favorably.

Keywords: Colorectal carcinoma, transcription factor, nuclear factor kappa B


How to cite this article:
Moorchung N, Kunwar S, Ahmed KW. An evaluation of nuclear factor kappa B expression in colorectal carcinoma: An analysis of 50 cases. J Can Res Ther 2014;10:631-5

How to cite this URL:
Moorchung N, Kunwar S, Ahmed KW. An evaluation of nuclear factor kappa B expression in colorectal carcinoma: An analysis of 50 cases. J Can Res Ther [serial online] 2014 [cited 2019 Sep 19];10:631-5. Available from: http://www.cancerjournal.net/text.asp?2014/10/3/631/139159


 > Introduction Top


Colorectal cancer (CRC) is a public health issue, contributing to 16,000 UK deaths per year, most of these occur in the elderly population. [1] The age adjusted incidence rates of CRC in all the Indian cancer registries are very close to the lowest rates in the world. [2] Population based time trend studies show a rising trend in the incidence of CRC in India. [3]

Nuclear factor kappa B (NFκB) is a transcription factor family, which belongs to the Rel family. [4] NFκB is considered to have an impact on many cellular phenomena such as inflammation, immune responses, proliferation, apoptosis, tumor progression and differentiation. [5] The stimuli that activate members of the Rel family are extensive and growing and it emphasizes their central role in transcriptional responses. [6] NFκB exists in the cytoplasm in the majority of cell types as homodimers or heterodimers of a family of structurally related proteins. [7] NFκB activation is associated with CRC. Colon cancer cell lines and human tumor samples, as well as nuclei of stromal macrophages in sporadic adenomatous polyps, were found to have increased NFκB activity. [8],[9]

NFκB activation has also been observed in many solid tumors, but so far no oncogenic mutations responsible for NFκB activation in carcinomas have been identified. However, through its ability to up-regulate the expression of tumor promoting cytokines, such as interleukin 6 or tumor necrosis factor α and survival genes, such as Bcl-XL, NFκB provides a critical link between inflammation' and cancer. [10]

This study was carried out to determine NFκB activation status in human colorectal carcinoma and its correlation to the histopathological grade and pathological stage of the tumor.

The objectives of the study were to:

  1. To study 50 cases of CRC and evaluate the grade and pathological stage
  2. To evaluate the degree of the p65 component of NFκB translocation into the nucleus of the cells
  3. To compare the benign and malignant areas with the degree of NFκB translocation
  4. To assess the grade and pathological stage with the degree of NFκB translocation.



 > Subjects and Methods Top


A total of 50 cases of colorectal carcinomas were evaluated. Small biopsies were not included. Only complete colorectal specimens were chosen. The specimens were grossed according to standard protocols. The hematoxylin and eosin (H and E) stain using Harris' H and E Y was used for staining the sections. All cases were reported according to the tumor node metastasis (TNM) system. The tumor and nodal status were recorded separately. The presence or absence of metastases was also noted.

For immunohistochemistry (IHC), sections were collected on poly L lysine coated slides. Primary antibody was monoclonal mouse anti-NFkB/p65 antibody (Santa Cruze) in a 1:200 dilution. A total of 500 epithelial cells and lymphocytes were enumerated in the benign and malignant areas. Areas which had stained intensely were evaluated. Evaluation was carried out under ×400 magnifications under the light microscopy and an average was taken. The results of the immunostaining were analyzed semi quantitatively as a percentage of positive cells.


 > Results Top


Demographic variables

Of the 50 cases, 29 were males (58%) and 21 were females (42%). The ages of the 50 patients ranged between 24 years and 85 years with a mean of 58.56 years. Of the 50 cases, 21 (42%) had involvement in rectum, 13 (26%) in the sigmoid colon, 1 (2%) in the transverse colon, 12 (24%) in the ascending colon and 3 (6%) in caecum.

Histopathological diagnosis and World Health Organization grade

Most of the cases were well-differentiated adenocarcinomas (58%). No cases of undifferentiated adenocarcinoma were seen [Table 1]. Most of the cases were of non-mucinous adenocarcinomas and the patients were predominantly in stage III [Table 2]. The histological types of the tumors in relation to the tumor grade are shown in [Table 3].
Table 1: The histopathological type and WHO grade of tumors


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Table 2: The histological type of the tumors and the TNM stage


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Table 3: Distribution of cases by relationship between the tumor grading and histological type of colorectal carcinoma (n=50)

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NFκB (p65 protein) expression by IHC

The grade of NFκB immunostaining was assessed in four compartments namely in the nuclei of the malignant epithelial cells, the nuclei of the benign epithelial cells, the lymphocytes in the malignant and the lymphocytes in the benign areas. 300-500 cells were assessed in each of these compartments and the grade of staining was expressed as a percentage. The grade of staining in each of these compartments was compared with the TNM stage of the tumor. The grade of staining was designated by the percentage of positive cells [Figure 1].
Figure 1: Immnunohistochemical stain for p65 in colon cancer tissues (a) non-neoplastic tissue. While weak immunoreactivity for p65 was detected in stromal cells including lymphocytes, reactivity was essentially absent in normal epithelial cells, ×100. (b) Lymphocytes staining with the antibody to nuclear factor kappa B (NFκB)/p65 (Santa Cruz) dilution 1 : 200, ×400. (c) Colon cancer staining with the antibody to NFκB/p65 (Santa Cruz) dilution 1 : 200, ×100. (d) Colon cancer staining with the antibody to NFκB/p65 (Santa Cruz) dilution 1 : 200, ×400. Note the presence of the nuclear stain

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Comparison of the malignant and the benign epithelial cells

It was found that the nuclei of the epithelial cells in the malignant areas showed a mean NFκB staining of 46.72% with a standard error of mean of 6.788%. The nuclei of the epithelial cells in the benign areas showed a mean NFκB staining of 11.59% with a standard error of mean of 3.711%. There was a significant difference between the degree of NFκB translocation in the epithelial cells of the benign and the malignant areas (P - 0.04) [Figure 2].
Figure 2: A correlation between the grade of nuclear factor kappa B staining in the epithelial cells in benign areas and the malignant areas

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Comparison of the grade of NFκB staining in the nuclei of the lymphocytes in the benign and malignant areas

The nuclei of the lymphocytes in the malignant areas showed a mean NFκB staining of 32.79% with a standard error of mean of 5.687%. The nuclei of the lymphocytes in the benign areas showed a mean NFκB staining of 13.50% with a standard error of mean of 3.597%. There was a significant difference between the degree of NFκB translocation in the lymphocytes of the benign and the malignant areas (P - 0.01) [Figure 3].
Figure 3: A correlation between the grade of nuclear factor kappa B staining in the lymphocytes in benign areas and the malignant areas

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Comparison of the grade of NFκB staining in the nuclei of the malignant epithelial cells with the TNM staging

There was a significant correlation between the grade of NFκB staining in the nuclei of the malignant epithelial cells with the tumor status (P - 0.001). There was also a significant correlation between the grade of NFκB staining in the nuclei of the malignant epithelial cells with the nodal status (P - 0.001). However, there was no correlation with age and sex [Figure 4] and [Figure 5].
Figure 4: A correlation between the grade of nuclear factor kappa B staining in the epithelial cells in malignant areas and the tumor status

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Figure 5: A correlation between the grade of nuclear factor kappa B staining in the epithelial cells in malignant areas and the nodal status

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Comparison of the grade of NFκB staining in the nuclei of the lymphocytes in the malignant areas with the TNM staging

There was a significant correlation between the grade of NFκB staining in the nuclei of the lymphocytes in the malignant areas with the tumor status (P - 0.001). There was also a significant correlation between the grade of NFκB staining in the nuclei of the lymphocytes in the malignant areas with the nodal status (P - 0.001) [Figure 6].
Figure 6: A correlation between the grade of nuclear factor kappa B staining in the nuclei of lymphocytes in malignant areas and the tumor and nodal status

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 > Discussion Top


CRC is a disease originating from the epithelial cells lining the colon or rectum of the gastrointestinal tract. Constitutive NFκB activation has been noted in many tumors, including CRCs. However, the precise role of this activation in CRC is unclear.

The result of the clinical parameters in our study was consistent with those of the previous studies as most cases were in older age group with male preponderance. The most common symptoms were local symptoms and rectum was the most common site of involvement (42%). [11],[12]

In our study, we showed that constitutive activation of NFκB was frequently observed in CRC. The nuclei of the epithelial cells in the malignant areas showed a mean NFκB staining of 46.72% and the nuclei of the lymphocytes in the malignant areas showed a mean NFκB staining of 32.79%. There was a significant correlation between the staining of nuclei of benign epithelial cells and malignant epithelial cells. There was also a significant correlation between the nuclei of lymphocytes in the malignant and the lymphocytes in the benign areas.

It has been seen that NFκB activation is associated with CRC. Colon cancer cell lines and human tumor samples, as well as nuclei of stromal macrophages in sporadic adenomatous polyps, were found to have increased NFκB activity. [13] The mechanism is not certain though apoptosis may have a role to play. NFκB activation is involved in apoptosis, although its role is not always straightforward. For instance, NFκB activation may lead to induction of apoptosis in some cell types. [14] This is perhaps because it, along with activator protein 1, can induce FasL expression. [15] NFκB can contribute to tumorigenesis in ways other than inhibition of apoptosis, e.g. by providing a direct stimulus toward proliferation via the activation of proto-oncogenes such as c-Myc and cyclin D1. [16] Thus, there are several ways by which NFκB can cause a malignancy to develop though it is difficult to say what has influenced the development of a malignancy in this study.

In our study, there was a significant correlation in the grade of NFκB staining in the nuclei of lymphocytes with the nodal status. We also found that epithelial nuclear positivity correlates well with the grade and stage of adenocarcinoma. This essentially means that NFκB staining correlates with a higher stage of the tumor. This is perhaps the first attempt to link NFκB levels with the grade and stage of the tumor. To date, NFκB has been studied extensively in breast cancer, prostate cancer preclinical and experimental models, but not as a clinical prognostic factor. [17] NFκB activation in breast cancer cell lines has been linked to the expression of both cyclin D1 and HER-2/neu, two well-known adverse outcome predictors in breast and possibly, prostate cancer. [18] The reasons for this association of NFκB staining and CRC are not clear but we offer a few possibilities.

At first, development of CRC is higher in patients affected by ulcerative colitis (UC) and to a lesser extent by Crohn's disease (CD) and the risk is directly proportional to the number of years of active disease. [19] It is likely that chronic inflammation may substantially contribute to cancer development. NFκB is known to activate the inflammatory pathways and it is likely that these cytokines released during inflammation and responsible for many of their proinflammatory effects, is responsible in promoting the growth of the colon tumors. In the present study, we did not find evidence of UC or CD in any of the specimens and therefore it is unlikely that inflammation has played a role in the pathogenesis of colorectal carcinoma in our series of cases. The second reason has been mentioned in a paper by Karin, [20] who has stated that constitutive activation of NFkB was frequently observed in colon cancer. It was suggested that this activation plays a key role in angiogenesis and antiapoptosis, promoting tumor growth. It is likely that NFκB activation acts through multiple pathways in influencing the progression of colorectal carcinoma.

We found that epithelial NFκB cytoplasmic positivity did not correlate with the grade or stage of the tumors This is due to the fact that NFκB in unstimulated resting cells is restricted to the cytoplasm bound to inhibitor kappa B (IκB) that subsequently prevents it from entering the nucleus. [21] When these cells are stimulated, specific kinases phosphorylate IκB causing its rapid degradation by proteosomes with release of NFκB and its passage into the nucleus where it influences activation of various cytokines and growth factors.

We conjecture at this point that translocation of NFκB to the nucleus in various compartments of mucosa is key event in pathogenesis of mucosal proliferation. If we could perhaps prevent activation of NFκB in colorectal carcinoma patients we could alter course of the disease favorably. Further research needs to be carried out on this concept to reap the therapeutic benefits.

 
 > References Top

1.
Quarini C, Gosney M. Review of the evidence for a colorectal cancer screening programme in elderly people. Age Ageing 2009;38:503-8.  Back to cited text no. 1
    
2.
Siegel R, Naishadham D, Jemal A. Cancer statistics, 2012. CA Cancer J Clin 2012;62:10-29.  Back to cited text no. 2
    
3.
Rastogi T, Devesa S, Mangtani P, Mathew A, Cooper N, Kao R, et al. Cancer incidence rates among South Asians in four geographic regions: India, Singapore, UK and US. Int J Epidemiol 2008;37:147-60.  Back to cited text no. 3
    
4.
Kojima M, Morisaki T, Sasaki N, Nakano K, Mibu R, Tanaka M, et al. Increased nuclear factor-kB activation in human colorectal carcinoma and its correlation with tumor progression. Anticancer Res 2004;24:675-81.  Back to cited text no. 4
    
5.
Caamaño J, Hunter CA. NF-kappaB family of transcription factors: Central regulators of innate and adaptive immune functions. Clin Microbiol Rev 2002;15:414-29.  Back to cited text no. 5
    
6.
Mercurio F, Manning AM. Multiple signals converging on NF-kappaB. Curr Opin Cell Biol 1999;11:226-32.  Back to cited text no. 6
    
7.
Shishodia S, Aggarwal BB. Nuclear factor-kappaB activation: A question of life or death. J Biochem Mol Biol 2002;35:28-40.  Back to cited text no. 7
    
8.
Greten FR, Eckmann L, Greten TF, Park JM, Li ZW, Egan LJ, et al. IKKbeta links inflammation and tumorigenesis in a mouse model of colitis-associated cancer. Cell 2004;118:285-96.  Back to cited text no. 8
    
9.
Yang N, Huang J, Greshock J, Liang S, Barchetti A, Hasegawa K, et al. NF-κB in cancer: From innocent bystander to major culprit. Nat Rev Cancer 2002;2:301-10.  Back to cited text no. 9
    
10.
Karin M. NF-kappaB as a critical link between inflammation and cancer. Cold Spring Harb Perspect Biol 2009;1:a000141.  Back to cited text no. 10
[PUBMED]    
11.
Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin 2011;61:69-90.  Back to cited text no. 11
    
12.
Siegel R. Colorectal Cancer Facts and Figures.  American Cancer Society Inc, Atlanta, GA- 30303-1002: American Cancer Society; 2011. p. 1-32.  Back to cited text no. 12
    
13.
Sartor RB. Mechanisms of disease: Pathogenesis of Crohn's disease and ulcerative colitis. Nat Clin Pract Gastroenterol Hepatol 2006;3:390-407.  Back to cited text no. 13
[PUBMED]    
14.
Miagkov AV, Kovalenko DV, Brown CE, Didsbury JR, Cogswell JP, Stimpson SA, et al. NF-kappaB activation provides the potential link between inflammation and hyperplasia in the arthritic joint. Proc Natl Acad Sci U S A 1998;95:13859-64.  Back to cited text no. 14
    
15.
Wang CY, Mayo MW, Korneluk RG, Goeddel DV, Baldwin AS Jr. NF-kappaB antiapoptosis: Induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation. Science 1998;281:1680-3.  Back to cited text no. 15
    
16.
Jo H, Zhang R, Zhang H, McKinsey TA, Shao J, Beauchamp RD, et al. NF-kappa B is required for H-ras oncogene induced abnormal cell proliferation and tumorigenesis. Oncogene 2000;19:841-9.  Back to cited text no. 16
    
17.
Ross JS, Jennings TA, Nazeer T, Sheehan CE, Fisher HA, Kauffman RA, et al. Prognostic factors in prostate cancer. Am J Clin Pathol 2003;120 Suppl: S85-100.  Back to cited text no. 17
    
18.
Zhou Y, Yau C, Gray JW, Chew K, Dairkee SH, Moore DH, et al. Enhanced NF kappa B and AP-1 transcriptional activity associated with antiestrogen resistant breast cancer. BMC Cancer 2007;7:59.  Back to cited text no. 18
    
19.
Viennois E, Chen F, Merlin D. NF-κB pathway in colitis-associated cancers. Transl Gastrointest Cancer 2013;2:21-9.  Back to cited text no. 19
[PUBMED]    
20.
Karin M. Nuclear factor-kappaB in cancer development and progression. Nature 2006;441:431-6.  Back to cited text no. 20
[PUBMED]    
21.
Oeckinghaus A, Ghosh S. The NF-kappaB family of transcription factors and its regulation. Cold Spring Harb Perspect Biol 2009;1:a000034.  Back to cited text no. 21
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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