Genomic sequence matters

Transcription factor NFκB is a key regulator of immune response. It also is constitutively active in many cancers as a major activator of anti-apoptotic gene expression that prolongs cancer cell survival by enhancing such features as proliferation, angiogenesis and metastasis.^1,2

NFκB is activated in numerous inflammation-driven cancers, e.g., colitis-associated colorectal cancer, hepatocellular carcinoma and lymphoma of mucosal-associated lymphoid tissue. Moreover, it also has a role in tumors such as thyroid carcinoma in which association with immune response is not obvious. NFκB is activated in all subtypes of human thyroid neoplasms. The activity of NFκB is particularly strong in poorly differentiated carcinoma. In thyroid cancer cell lines it correlates with resistance to drug-induced apoptosis.³ In undifferentiated thyroid carcinoma, activation of NFκB by tumor necrosis factor alpha (TNF-α) led to cytomorphologic differentiation of cancerous cells, and induction of thyroid-specific secretion of thyroglobulin.⁴

The influence of microRNAs, especially miR-155 and miR-146, on the immune response has been extensively studied.⁵ That miR-146a might be up-regulated by NFκB was recently shown.⁶ NFκB induces the expression of miR-146a upon ligation of toll-like receptors (TLR2, TLR4 or TLR5) as well as stimulation by TNF-α or interleukin 1 β.^7–9 It is suggested that miR-146a serves as an important negative regulator of the TLR and NFκB signaling pathway as the IL-1 receptor-associated kinase 1 (IRAK1) and TNF receptor-associated factor 6 genes (TRAF6) were proved to be direct targets of miR-146a.⁶ Thus, microRNA-146a seems to function as a pro-apoptotic molecule by inhibiting the NFκB pathway and blocking its impact on cell proliferation, tumor-progression and cancer cell survival. When expressed in the highly metastatic human breast cancer cell line MDA-MB-231 microRNA-146a leads to impaired NFκB activity. Overexpression of miR-146 through inhibition of the NFκB pathway led to impaired invasion and migration in vitro and suppressed experimental lung metastasis in mice.¹⁰

MiR-146 has been reported to be highly expressed in breast, pancreatic, ovarian, stomach and thyroid cancer. In papillary thyroid carcinoma (PTC) miR-146a is up-regulated 19 fold¹¹ and its expression is dependent on a sequence variation.¹² A single nucleotide polymorphism (SNP) within pre-miR-146a (rs2910146; G/C) seems to disrupt the pro-apoptotic function of the miR by decreasing the total amount of mature miR-146a with consequent reduced inhibition of miR target genes such as IRAK1 and TRAF6.^12,13 In an association study of 608 PTC patients and 901 controls, the GC heterozygous state was associated with an increased risk of acquiring PTC (odds ratio = 1.62, p = 0.000007), whereas both homozygous states (GC and CC) were protective with odds ratio = 0.42 for the CC genotype (p = 0.003) and odds ratio = 0.69 for the GG genotype (p = 0.0006). Moreover, 4.7% of informative tumors had undergone somatic mutations of the SNP sequence probably as a step in the clonal selection during carcinogenesis.¹² Heterozygosity as a genetic risk rather than either homozygosity is a rare phenomenon (referred to as overdominance) and should be critically evaluated. This led to the observation that the passenger strand of the pre-miR produces mature miR. Thus, in GC heterozygotes totally 3 mature miRs were produced (main strand; passenger strand G, passenger strand C), each with distinct target genes.¹⁴

To begin to unravel the functional consequences on target genes a microarray expression analysis was performed on thyroid tumors from 16 PTC patients who were either GG or GC, revealing significantly different transcriptomes. In particular, the genes involved in regulation of apoptosis were differentially transcribed in heterozygotes compared to GG homozygotes (44 genes, p=0.0001). Moreover, among genes differentially regulated in heterozygotes 12 genes were designated “positive regulators of NFκB” (p=0.009).¹⁴

In unstimulated cells, NFκB is retained in the cytoplasm by specific inhibitors, the IκB proteins. Stimulation with the proinflammatory cytokines, TNF-α or IL-1, induces IκB phosphorylation and ubiquitin-dependent proteasomal degradation resulting in nuclear entry of NFκB dimers to initiate target gene transcription. IκB phosphorylation is catalyzed by the IκB kinase (IKK) complex.¹⁵ In the study described above,¹⁴ numerous genes leading to activation of NFκB were up-regulated in heterozygotes, as were many genes targeted by NFκB including the proinflammatory chemokines IL8 and CFLAR (Table 1). Caspase 4 and caspase 8 were also up-regulated; however in the presence of induced CFLAR the latter was most likely inactive.¹⁴

Table 1.

Impact of the miR-146a polymorphism (GC vs. GG) on the expression of genes involved in the NF-κB pathway and in the regulation of apoptosis

Gene	Name	Fold change	P value
LITAF	Lipopolysaccharide-induced TNF factor	1.38	0.014
Fas	TNF receptor superfamily, member 6	1.86	0.015
TNFRSF21	Tumor necrosis factor receptor superfamily, member 21	1.89	0.022
S100A8	S100 calcium binding protein A8	3.50	0.004
IL3RB	Colony stimulating factor 2 receptor, beta, low-affinity	1.90	0.026
IL1R1	Interleukin 1 receptor, type 1	1.55	0.006
BCL10	B-cell CLL/lymphoma 10	1.27	0.021
IKBKB	Inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase beta	1.38	0.043
IL8	Interleukin 8	2.32	0.026
CFLAR	CASP8 and FADD-like apoptosis regulator	1.35	0.016
CASP4	Caspase 4	1.45	0.021
CASP8	Caspase 8	1.40	0.023

Thus, these results suggest significantly higher activation of the NFκB pathway in GC heterozygotes of miR-146a as compared to GG homozygotes. We speculate that this phenomenon might promote tumor progression and be responsible for the increased risk of PTC in heterozygotes.

A possible stimulus for an induction of the NFκB pathway in thyroid might be ionizing radiation, the strongest environmental risk factor for thyroid cancer. Ionizing radiation is also used for the treatment of thyroid cancer. It induces DNA damage, e.g. double strand DNA breaks, and leads to significant activation of p53 and apoptosis. NFκB is rapidly activated in the course of an immediate early response of thyroid cancer cells to ionizing radiation leading to increased thyroid cell survival. In an undifferentiated thyroid cancer cell line inhibition of NFκB increased radiosensitivity and enhanced ionizing radiation-induced apoptosis.¹⁶ Cells that do not undergo apoptosis after ionizing radiation are prone to genetic instability, increased rate of mutation, and accelerated cancer evolution. We suggest that thyroid cells heterozygous at miR-146a present higher activity of NFκB and lower potential of inhibition of this pathway and therefore are more likely to survive after ionizing radiation.