NF-kappa B expression in resected specimen of colonic cancer is higher compared to its expression in inflammatory bowel diseases and polyps

There is growing evidence linking inflammation, cancer development, and nuclear factor kappa B (NF-κB)1.

Preclinical studies using animal models have shown that NF-κB is involved in colorectal carcinogenesis. In mice models, the NF-κB pathway has been directly linked to intestinal inflammation and to development of colitis-associated cancer16,17,18. In mice models of CRC, NF-κB has also been linked to progression of tumor growth17,19.

Yet, the clinical implications of NF-κB in human inflammatory or cancer diseases of the colon have rarely been studied and evidence for its relevance to these patients is limited20.

To date, activating mutations of NF-κB in CRC have not been reported3. Nonetheless, constitutive activation of NF-κB has been observed in human CRC and associated with higher tumor stage18,21, treatment resistance19,22 and poor survival outcomes3. In addition, activation of the NF-κB main protein p65 in colon of metastatic CRC patients correlated with liver metastasis and poor clinical outcomes, measured as overall survival; suggesting it may have prognostic value in this disease17,23.

In this study, we evaluated the levels of RelA/p65 protein, which is a key in the canonical pathway of NF-κB activated by tumor necrosis factor alpha, Toll-like receptor ligands, and interleukin-15. RelA/p65 is less involved in the non-canonical pathway, activated by other ligands and signals. Via the canonical pathway, different combinations of subunits with RelA/p65 protein are shown to activate this signaling pathway, regardless of the specific dimer formed5,6.

RelA/p65 protein was chosen based on the intent to compare protein expression between inflammatory conditions of the colon and those of cancerous conditions. In other words, since this is a basic proof of concept study we went for a gross evaluation and looked for the wide picture.

Nonetheless, here we evaluated the expression levels of RelA/p65 NF-κB protein in colonic tissues of patients undergoing surgery for various etiologies.

We have found that NF-κB levels were significantly higher in CRC cancer compared to tumor-free margins and to benign tissues, including IBD specimens. We consider this novel finding to be unpredicted since the role of the NF-κB signaling in inflammation is well-established compared to its involvement in CRC. Thus, we did not anticipate NF-κB levels to be significantly higher in CRC compared to tumor-free IBD specimens. Our findings stressed the important role of NF-κB in colorectal cancer.

Additionally, we have found that the expression of NF-κB in adenomatous polyps was similar to its expression in IBD tissues. This finding may support the hypothesis that NF-κB plays an important role early in the process of colonic dysplasia development that may lead to cancer. Interestingly, this finding correlates to other studies linking between NF-κB and colitis-associated adenoma development24.

Some studies tried to look for an association of NF-κB to cancer aggressiveness. Pyo et. al. investigated the correlation between phosphorylated NF-κB (pNF-κB, the activated form of the NF-κB protein) nuclear expression in pathological tissues and clinicopathological characteristics of CRC patients20. That study included 261 patients. They have demonstrated that pNF-κB was significantly correlated with frequent perineural invasion, lymph node metastasis and higher disease stage. The relation to disease stage was attributed to the association with positive lymph nodes but not to the tumor T stage, which was not correlated with pNF-κB levels. These findings are comparable in some aspects to our findings. Interestingly, in our study, NF-κB expression in the margins of resection was associated with positive node status of CRC patients. This may lead to the assumption that the associated processes that link NF-κB with colorectal cancer is related to the entire colonic tissue and not only to the tumor site itself. In our study, we could not show a statistical significance levels of NF-κB expression in the tumoral tissue itself between negative and positive nodes status, however, this may be related to the low number of patients we had in these groups.

Nevertheless, based on our results and others20, this indicates that NF-κB signaling may be involved in the dissemination processes of colonic adenocarcinoma cells. But, as its levels have not correlated with the extent of tumor involvement (T stage), we believe that NF-κB is more involved in the tumor metastatic process rather than direct invasiveness.

This data supports the hypothesis on the role of NF-κB in colorectal carcinogenesis, underlining the point that this signaling pathway may be more significant when involved in colon cancer dissemination mechanisms than in the primary colonic carcinogenic process.

In our study 4 out of 30 patients have developed recurrence. We could not show a statistical significant difference in the expression NF-κB when comparing these patients with patients that had no recurrence despite a numerical difference (Fig. 5). We believe that this may be related to the low number of patients in our series that does not allow a reliable statistical evaluation.

To further assess this hypothesis, we suggest a long-term, prospective clinical study measuring NF-κB expression levels of CRC patient colonic specimens matched with long-term follow-up data regarding metastatic disease recurrence.

Furthermore, our data revealed that NF-κB tumor levels positively correlated with expression in tissue margins (Fig. 1), which demonstrates that the activation of this signaling pathway in colons with pathological tissue is diffuse and not limited to the tumor mass. This interesting finding may suggest either a predisposition to increased NF-κB expression among patients with colonic pathologies or a remote effect of the primary lesion on the adjacent normal colonic mucosa. This also supports the concept that NF-κB may affect the metastatic potential of the tumor to its surrounding tissue more broadly than the tumor itself does.

As activation of NF-κB pathways has been previously associated with poor prognosis, it is possible that members of the NF-κB pathways could serve as prognostic markers or novel therapeutic targets for CRC. Indeed, several suggestions for anti-NF-κB therapeutics for colon cancer treatment have been examined in pre-clinical trials [reviewed in 5,25]. But despite the promising results, no clinical trials have been published to date. Thus, there is a lack of clinical evidence to validate this potential.

Another point regarding the association of NF-κB with poor prognosis is its role in cancer resistance to therapy. NF-κB activation has been suggested to be associated with resistance to therapy in gastrointestinal malignancies, mainly in predicting resistance to chemoradiation in esophageal cancer26 and to a combination of irinotecan and cetuximab in CRC27. Neither of these studies, however, addressed the possibility that NF-κB is a prognostic rather than a predictive clinical marker. Furthermore, the literature on the role of NF-κB in CRC progression is limited [reviewed in 5 and 25].

One of the limitation of our study is the lack of evaluation of other important biomarkers that are known to activate or be activated by NF-κB.

Two reviews on the association between the inflammation process of IBD and the development of colorectal cancer have stressed the relation between certain cytokines such as IL-6, IL- α and TNF-α to NF-κB and proposed that its mechanism of action is related to its ability to block apoptosis by positively regulating the expression of anti-apoptosis proteins28,29.

Our study did not aim to explore the mechanism of action of NF-κB but to demonstrate another evidence from a different angle for its pivotal role in the development of colorectal cancer.

Our study is novel in its methods (quantifying the immunochemistry staining in a relatively new method) and in its idea (comparing expression of NF-κB between cancer and IBD).

Adding a western blot and Qpcr could have further verifying the expression of NF-KB, however, the technique that we have used to quantify NF-KB is a well-established and reliable one13,14.

The results further support the accumulating data on the important role of NF-κB in CRC and should encourage other researchers to use this method and to further investigate the expression of NF-κB in IBD patients with and without cancer.

We have added in our study another evaluation of a biomarker, CEA. CEA is not directly related to NF-Κb and thus We did not expect to find a correlation between CEA and NF-KB. however, CEA is known to be highly associated with both CRC and IBD. Thus, the difference that we have found between CEA expression in cancer tissue compared to IBD that goes along with the difference in NF-κB expression supports the validity of our methods and findings.

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