Somatic mutation of TRAF3 gene is rare in common human cancers and acute leukemias

To the Editor

In canonical NF-κB pathway, activation of trimeric IκB kinase (IKK) complex phosphorylates and degrades IκB, and subsequently releases p50:RelA and p50:cRel dimmers. In non-canonical NF-κB pathway, activated NF-κB-inducing kinase (NIK) in association with IKKα binds to p100, leads to p100 processing to p52 and release p52:RelB dimmers [1], [2]. These dimmers act as transcription factors that regulate the expression of many genes. The NF-κB pathways are also regulated by many other proteins, including TRAF2, TRAF3, CYLD, cIAP1, cIAP2 and CD40 [1], [2]. NF-κB is constitutively active in most tumor cell lines and cancer tissues, whereas it is rarely activated in normal cells except for proliferating immune cells and astrocytes [1], [2]. Suppression of NF-κB activation inhibits proliferation, leads to cell cycle arrest, and causes apoptosis, strongly suggesting roles of NF-κB activation in tumorigenesis [1], [2].

Recently, two research groups identified that genetic and epigenetic alterations of genes encoding proteins in the NF-κB pathways resulted in constitutive activation of NF-κB in multiple myeloma (MM) cell lines and patient samples [3], [4]. Among the alterations, TRAF3 mutation was the most common event. Functionally, TRAF3 inactivation enhances NF-κB signaling and cell survival in MM cells. Because NF-κB signaling is constitutively activated not only in MM but also in acute leukemias and solid cancers [1], [2], it could be hypothesized that TRAF3 gene mutation could be responsible to the NF-κB activation in other cancers besides MM as well. To explore this issue, in the present study, we analyzed TRAF3 mutations in common solid cancers and acute leukemias.

For this, we analyzed the TRAF3 gene in methacarn-fixed tissues of 47 gastric adenocarcinomas, 47 colorectal adenocarcinomas, 47 breast invasive ductal carcinomas, 47 hepatocellular carcinomas and 47 non-small cell lung cancers, and fresh non-fixed fresh tissues of 37 acute myelogenous leukemias (subgroup details available on request) by a polymerase chain reaction (PCR)- single strand conformation polymorphism (SSCP) assay. All of the patients of the cancers were Koreans. In solid tumors, malignant cells and normal cells were selectively procured from hematoxylin and eosin-stained slides using a 30G1/2 hypodermic needle (Becton Dickinson, Franklin Lakes, NJ) affixed to a micromanipulator, as described previously [5], [6]. DNA extraction from the microdissected tissues was performed by a modified single-step DNA extraction method by proteinase K treatment [5], [6]. Genomic DNA each from tumor cells and normal cells from the same patients were amplified by PCR with 14 primer pairs covering entire coding region (exon 2-11) of human TRAF3 gene. Radioisotope ([³²P]dCTP) was incorporated into the PCR products for detection by autoradiogram. The PCR products were subsequently displayed in SSCP gels. Other procedures of PCR and SSCP analysis were performed as described previously [5], [6].

On the SSCP autoradiograms, all of the PCR products from the cancers were clearly seen. However, the SSCPs from them did not reveal any aberrantly migrating band compared to wild-type bands from the normal tissues, indicating there was no evidence of somatic mutations of the TRAF3 in the cancers. To confirm the SSCP results, we repeated the experiments twice, including tissue microdissection, PCR and SSCP to ensure the specificity of the results, and found that the data were consistent.

As a possible mechanism of NF-κB activation in common human cancers, we analyzed TRAF3 mutation in gastric, colorectal, lung, hepatocellular and breast carcinomas, and acute myelogenous leukemias. However, we detected no TRAF3 mutation in the cancer specimens. Compared to the high incidence of TRAF3 mutation in the MM (up to 19.4%) [3], [4], the TRAF3 incidence in each of the cancers analyzed in this study was significantly low (Fisher's exact test, p < 0.05). Our data indicate that the NF-kB activation frequently observed in gastric, colorectal, lung, hepatocellular and breast carcinomas, and acute myelogenous leukemias may not be a result of TRAF3 mutation in the cancer cells. In addition to the TRAF3, alterations of NF-κB-related genes in the MM included NIK (amplification and translocation), NF-κB2 encoding p52/p100 subunit (point mutation), NF-κB1encoding p105 subunit (amplification), CYLD (deletion and point mutation), CD40 (overexpression), cIAP1 (deletion) and cIAP2 (deletion) [3], [4]. To identify mechanisms of NF-κB activation in solid cancers and leukemias, alterations of these genes should be further analyzed in future studies.

Acknowledgements

This work was supported by the funds from KOSEF (R01-2008-000-10014-0).