Brief communicationRelative abundance of full-length and truncated FOXP1 isoforms is associated with differential NFκB activity in Follicular Lymphoma
Introduction
Follicular Lymphoma (FL) is the most common type of indolent non-Hodgkin's Lymphoma (NHL) in adults. Despite recent advances, the disease remains incurable [1]. The FL International Prognostic Index (FLIPI) uses clinical criteria to stratify patients into prognostic groupings, but there remains a large amount of heterogeneity in disease outcome within FLIPI sub-categories. In order to more accurately predict the clinical course of FL, molecular markers that relate to disease behaviour need to be elucidated and their role defined. One potential candidate is FOXP1, a transcriptional repressor belonging to the FOX family of forkhead box/winged-helix domain-containing proteins. It is expressed in the majority of FL tumors [2], and strong immunohistochemical (IHC) staining for FOXP1 has been associated with poor outcome in another common type of NHL, Diffuse Large B-cell Lymphoma (DLBCL) [3], [4]. However, the role of FOXP1 has not yet been investigated in FL.
FOXP1 has been described as both a tumor suppressor candidate, due to deletion of its locus in multiple solid tumor types [5], [6], as well as a potential oncogene, due to its over-expression in other tumors sometimes associated with genetic translocation [7]. Investigations into FOXP1 have yielded some interesting speculations regarding its role in malignancies. FOXP1 is known to repress transcription of some NFAT- and NFκB-responsive genes, such as IL-2, which contain forkhead (FKH) consensus sequences in their promoter region proximal to NFκB binding sites [8]. However, 7 NH3-terminally truncated isoforms of FOXP1 have recently been described [9], 3 of which were found to be expressed in DLBCL samples. Truncated isoforms were particularly associated with a subtype of DLBCL that is characterised by constitutive NFκB activity [9], suggesting that they may have an inhibitory effect on the repressor activities of full-length isoforms and promote NFκB signalling. This proposed alternate role for full-length and truncated FOXP1 isoforms is in line with observations that have described FOXP1 as both a tumor suppressor gene and an oncogene [10].
Immunohistochemical (IHC) analysis of FOXP1 employs the JC12 antibody, which is unable to differentiate between isoforms. We have therefore developed a novel quantitative real-time PCR (qRT-PCR) assay to measure the relative abundance of full-length and truncated isoforms of FOXP1 at the transcript level. We have used this assay to measure the relative abundance of FOXP1 isoforms in tumor samples from a cohort of FL patients and compared measurements to those derived from control hyperplastic lymphoid tissue (HLT). These measurements were then correlated with microarray-based expression measurements of NFκB-associated genes. Using this approach we found that the FL tumors have increased relative abundance of truncated isoforms of FOXP1 compared to control HLT, and this correlated with the transcript abundance of a large number of NFκB-associated genes. This suggests that FOXP1 may function in regulating NFκB activity in FL.
Section snippets
Methods
Diagnostic fresh-frozen lymph node specimens from 20 FL patients (9 Grade I, 6 Grade II and 5 Grade III) were obtained from the Australian Leukemia and Lymphoma Group Tissue Bank and Bio Options bio-repository service (Los Angeles, CA). For use as control HLT, tonsil tissue was obtained from 12 cancer-free tonsillitis patients. DNA and total-RNA were extracted from each sample (see Supplementary Methods), and RNA integrity was analysed with an Agilent Bioanalyser 2100 using the NanoChip
Results
The effect of isoform-2 transcripts on N-terminal assay measurement, and hence the ability of FOXP1ΔCT values to truly reflect the relative abundance of full-length and truncated isoforms, was assessed by quantification of isoform-2 in all FL and control HLT samples. FOXP1 isoform-2 was detected at low levels in all control HLT samples, with isoform-2 ΔCT values ranging between 16.15 and 20.46 (mean = 17.85, SD = 1.31). In contrast, 50.0% of FL samples had undetectable levels of isoforms-2, and
Discussion
The N-terminal region of FOXP1 has been proposed to act as a complex docking site for transcriptional co-repressors, such as carboxyl-terminal binding protein-1 (CtBP-1) [11]. Truncated isoforms of FOXP1 not only lack their N-terminal coiled-coil domain, which may mediate such interactions, but also lack most of or the entire second poly-glutamine domain—the length of which has been shown to correlate with transcriptional repressor activity [8]. The loss of function associated with N-terminal
Acknowledgements
We would like to acknowledge the Australasian Leukaemia and Lymphoma Group Tissue Bank for the provision of Follicular Lymphoma samples for this project. The ALLG Tissue Bank is supported by Pricewaterhouse Coopers, the Leukaemia Foundation of Australia and National Health and Medical Research funding. This research was supported by funding from the Griffith Medical Research College and a Herbert Family philanthropic scholarship.
References (12)
- et al.
Follicular lymphoma: time for a re-think?
Blood Rev
(2005) - et al.
Strong expression of FOXP1 identifies a distinct subset of diffuse large B-cell lymphoma (DLBCL) patients with poor outcome
Blood
(2004) - et al.
Multiple domains define the expression and regulatory properties of Foxp1 forkhead transcriptional repressors
J Biol Chem
(2003) - et al.
Potentially oncogenic B-cell activation induced smaller isoforms of FOXP1 are highly expressed in the activated B-cell-like subtype of DLBCL
Blood
(2008) - et al.
The FOXP1 transcription factor is expressed in the majority of follicular lymphomas but is rarely expressed in classical and lymphocyte predominant Hodgkin's lymphoma
J Mol Histol
(2005) - et al.
The FOXP1 winged helix transcription factor is a novel candidate tumor suppressor gene on chromosome 3p
Cancer Res
(2001)
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