AID expression increased by TNF-α is associated with class switch recombination of Igα gene in cancers

Recently, immunoglobulins (Igs) were unexpectedly found to be expressed in epithelial cancers. Immunoglobulin class switching or class switch recombination (CSR) is a natural biological process that alters a B cell’s production of antibodies (immunoglobulins) from one class to another. However, the mechanism of CSR of Ig genes in cancer is still unknown. Here, we confirmed by detecting the hallmark of CSR that the Igα gene in cancer underwent CSR. Then we focused on activation-induced cytidine deaminase (AID), a crucial factor for initiating CSR. Further studies using tumor necrosis factor (TNF)-α stimulation and specific inhibitor of NF-κB revealed that TNF-α could increase AID expression through NF-κB signaling. Finally, we demonstrated that AID could co-localize with protein kinase A and bind to the switching (Sα) region of the Igα gene. Overexpression of AID obviously enhanced Igα heavy chain expression and its binding ability to the Sα region. These findings indicated that TNF-α-induced AID expression is involved with CSR in cancer.


INTRODUCTION
The only resource of immunoglobulin (Ig) molecules has traditionally been believed to be from B lymphocytes. In humans, all cells include germ line (GL) Ig genes, which comprise several V, D, J, and C genes. During maturation and differentiation of B lymphocytes, the Ig genes undergo V(D)J recombination, class switch recombination (CSR), and somatic hypermutation (SHM), and only then can immunoglobulins be expressed. In order to produce variable genes of Igs, V(D)J recombination assembles different V, (D), and J regions randomly, which play important roles in recognizing different agents. Class switching combines a downstream constant region, such as the Ca or Cc region, to the V(D)J region, converting an Ig molecule from IgM to IgA or IgG.
We previously cloned a transforming Tx gene from the gDNA library of the CNE2 nasopharyngeal carcinoma cell line, and this Tx gene was then determined to be an abnormal Igk gene lacking variable regions. 1 This innovative discovery suggested the possibility of Ig expression in cancer cells and several research groups categorically confirmed abnormal expression and secretion of Igs in non-lymphoid cancer cells. 2,3 Scientific researchers have demonstrated the biological functions [4][5][6] and the molecular mechanisms of Ig expression in cancer, [7][8][9][10] including the basis of V(D)J recombination. 11,12 However, whether Ig genes undergo class switching has not been determined and the mechanisms underlying cancer cell initiation of CSR are still unknown.
CSR of immunoglobulins occurs in mature B cells in response to foreign antigen stimulation and co-stimulatory signals. Mature B cells can express different classes or isotypes of Ig only after undergoing CSR, which assists the antigen-presenting cells (APC) in eliminating pathogens. The class or isotype of immunoglobulin is determined by the heavy chain constant (C H ) region, and different C H regions have different affinities for cell surface receptors, such as the Fc receptor, poly-IgA receptor and complement, thus determining the immunoglobulins' effector functions. 13 Two pivotal factors are required for the initiation of CSR, including GL transcription of the unrearranged constant region and the B cell-specific activation-induced cytidine deaminase (AID). GL transcription and AID expression can be activated by antigen and cytokine stimulation. Once activated, AID is phosphorylated by protein kinase A (PKA) at Ser38 and Thr27, which is required for the binding of AID by replication protein A (RPA).The interaction between AID and RPA can increase the binding ability of AID with DNA. 14 Switching regions are composed of tandemly repeated sequences that are located in the introns upstream of each C H region gene, except C d . Clearly, AID targets pentameric sequences (GAGCT and GGGCT) in switching (S) regions and preferentially deaminates the dC to dU nucleotides. 15,16 Then UNG and APE are specifically recruited to sites of AID activity and excise the dU residue and phosphate backbone, producing single-strand breaks (SSBs). 17,18 When SSBs appear, converting SSBs to double-strand breaks (DSBs) is required for CSR. After formation of the DSBs in the donor and acceptor S regions, the two S regions are recombined by performing nonhomologous end-joining, completing the CSR. 13 Due to the crucial role of AID in CSR, understanding how AID is regulated is extremely important. Four regulatory regions are involved in transcription when comparing nucleotide sequences around the AID locus. The most important region is located upstream of the transcription start site (TSS), containing the AID promoter and binding sites for several transcription factors, such as HoxC4-Oct, NF-kB and STAT6. 19,20 Transcription factor HoxC4 is preferentially expressed in germinal center B cells, and is upregulated by lipopolysaccharide (LPS) and interleukin (IL)-4. The NF-kB binding site is suggested to be responsive to viral infection and tumor necrosis factor (TNF)-a signaling. 21 Moreover, NF-kB, STAT6, and the Smad proteins have been reported to play key roles in transactivating AID expression in B cells. 20 The signaling pathway that stimulates the activation of these cytokines is deeply involved in a variety of inflammatory responses associated with carcinogenesis in epithelial organs. In humans, AID is abundantly expressed in Epstein-Barr virus (EBV)-positive Burkitt's lymphoma cells. This is probably due to the expression of latent membrane protein (LMP1) on the cell surface, which mimics continuous stimulation of CD40 signaling. More recently, many tumor-causing viruses have been shown to induce AID expression in both B and non-B cells. AID is frequently expressed in adult T cell leukemia caused by human T-cell leukemia virus (HTLV)-1 infection. 22 The hepatitis C virus (HCV) induces AID through NF-kB signaling in hepatocellular carcinoma. 23,24 Further, Helicobacter Pylori, a gastric cancer-causing agent, also induces AID in the gastric epithelium through the IKK-b-dependent NF-kB activation pathway. 25 Together, these findings support the hypothesis that inflammatory stimulation of epithelial cells could induce aberrant AID expression and initiate and/or promote oncogenic signaling by enhancing susceptibility to mutagenesis.
In this study, we first demonstrated that Ig genes undergo CSR by detecting the hallmark of CSR at the genomic and transcriptional levels, and found that the integrated Iga gene, VDJ-Ca, and the looped-out circle Sa-Sm gene are present in cancer cells. To elucidate the molecular mechanism of class switching, we focused on AID, a crucial factor associated with CSR in B lymphocytes. We verified that AID is abnormally expressed in NPC tissues and several cancer cell lines. Further studies indicated that TNF-a could play an important role in AID expression through NF-kB signaling. Moreover, we used by immunofluorescence-confocal assays to confirm that AID can interact with PKA. We used chromatin immunoprecipitation (ChIP) assays to verify that AID and PKA could target to the Sa region of Ig genes, which is essential for Iga expression. These results indicated that the unexpected expression of AID induced by TNF-a is associated with CSR of Ig genes in cancer.

Transfection and plasmids
Cancer cells were cultured overnight in 6-well plates at a density of 1 3 10 6 cells per well and were transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Each transfection contained 2.5 mg/well of the pMSCV-GFP-AID overexpression plasmid (Addgene, Cambridge, MA, USA) or the pMSCV-GFP control plasmid. Cells were harvested at 24 h after transfection and lysates were analyzed by Western blot.

RT-PCR and nested PCR
Total RNA was isolated from different cancer cell lines and immortalized cells, using the Trizol reagent (Invitrogen) according to the manufacturer's instructions. cDNAs were synthesized with the SuperScript II Kit (Invitrogen), and the resulting cDNA was subjected to RT-PCR. Total gDNA was isolated from different cells using a DNA extraction kit (Qiagen, Dusseldorf, Germany) according to the manufacturer's instructions. Nested PCR using gDNA was performed with two rounds of the PCR reaction. The first round of PCR produced a long-length product, and the second round produced a shorter length product. Nested PCR shows greater specialty and sensitivity. Most of primers used for RT-PCR and nested PCR are previously reported. 26,27 PCR products TNF-a upregulates AID expression via NF-kB signaling Z Duan et al.
were separated on 2% agarose gels and visualized with SYBR safe (Invitrogen).

Protein extraction
Cells were harvested, washed twice with ice-cold phosphatebuffered saline (PBS) and disrupted for 30 min in immunoprecipitation (IP) lysis buffer (Pierce, Rockford, IL USA) containing a protease inhibitor cocktail (Roche, Basel, Switzerland). The mixture was then centrifuged at 15 000g for 15 min after sonication. The supernatant fractions were collected as whole cell lysates and used for Western blot assays. Protein concentration was determined with the BCA Assay Reagent (Pierce).

Immnunohistochemistry analysis of an NPC tissue array and NPC biopsies
The NPC tissue array was purchased from Pantomics (Richmond, CA, USA). The slides are placed in a 60uC oven for 30 min followed by a xylene bath. Two changes of xylene and different concentration of ethanol were performed for 5 min each to remove paraffin and rehydrate the tissue. This is followed by quenching endogeneous peroxidase activity with 0.15% hydrogen peroxide in methanol. The slides were then incubated with an rabbit anti-human AID monoclonal   Chromatin immunoprecipitation assay Cells in the logarithmic growth phase were subjected to ChIP assays as previously described. 10 Antibodies included rabbit anti-AID (Abcam), anti-PKA (Cell Signaling, Danvers, MA, USA) or normal rabbit IgG.

Statistical analysis
All statistical calculations were performed using the statistical software program SPSS (ver.12.0). Differences between various groups were evaluated by the Student's t-test. The difference was statistically significant when p , 0.05 or p , 0.01.

RESULTS
The hallmark of CSR are present in multiple epithelial cancers Class switching is accompanied by deletion of circular DNA from the Ig heavy chain locus 28 ( Figure 1A). To determine whether Ig genes undergo CSR in cancer, we first detected the VDJ-C H transcript and the Ig I H -C H GL transcript in several cancer cell lines. Results showed that the VDJ-Cm, VDJ-Ca, VDJ-Cc transcripts are present in various cancer cell lines ( Figure 1C), suggesting that CSR has been completed in these cancer cell lines. Ig GL transcription is a critical factor for initiating CSR. Results also indicated that Ig I H -C H GL transcripts  including Ig Im-Cm, Ig Ia-Ca and Ig Ic-Cc are produced in these cells ( Figure 1D). The best marker of active CSR should be expressed specifically during CSR and disappear quickly after CSR has been completed. A resultant circular DNA could be a good marker for active CSR. 28 However, PCR amplification of circular DNA has limited sensitivity, because only a single copy of circular DNA is generated in each switched cell. We therefore used nested PCR, which is more specific and sensitive, to amplify the reciprocal switch junction Sa-Sm, from the gDNA of various cancer cell lines. Two pairs of primers were designed for the amplification of the junctions of circular DNA (Figure 1B), and the results revealed that circular DNA Sa-Sm is present in multiple cancers ( Figure 1E). We then determined whether the I H promoter, which is activated by cytokine stimulation before CSR, remains active even on circular DNA. We performed RT-PCR to detect the transcript of this junction. Results showed that the Ia promoters was still active, leading the transcription of Ia-Sm ( Figure 1F). We confirmed the PCR products by sequencing (data not shown). These findings indicated that Ig genes have undergone CSR in various cancer cell lines.

AID is unexpectedly expressed in cancer
Both Ig GL transcripts and AID are key regulators of CSR. The expression of AID is found in B lymphomas, in myeloid leukemia, and in pathogen-induced tumors such as adult T cell leukemia. Here we determined whether AID is expressed in NPC tissues, using lymphoma tissue as a positive control. Immunohistochemistry results indicated that both chronic inflammation tissue and NPC tissue expressed AID abnormally, but the expression level of AID in NPC was much higher than that in chronic inflammation tissue (Figure 2A and B). in the NP69 immortalized cell line ( Figure 2C and D). Furthermore, we also detected AID expression in other immortalized cell lines, including NP460, HBE, HaCaT, and HUVE-12 ( Figure 2E). Further study using immunofluorescence showed that AID is mainly located in the nucleus of CNE1 cells ( Figure 2F). Notably, AID localizes differently in tissue and in cells and AID localization in the nucleus has also been reported by others. 29 These results indicated that AID is unexpectedly expressed in epithelial cancer cells.

TNF-a induces AID expression through NF-kB signaling in cancer
In B lymphocytes, AID expression can be induced by TNF-a stimulation. To explore whether TNF-a can affect AID expression (c-d) Western blot assay results indicate that overexpression of AID decreases Iga heavy chain expression. Expression level for AID or Iga was estimated by densitometry and was presented as a ratio to the respective loading control. (e-f) Overexpression of AID enhances the binding ability to the Sa region of Ig genes. The binding ability was also confirmed by real-time PCR after ChIP assays. (g-h) When transfected with AID shRNA to knockdown AID expression, cancer cells express attenuated Iga heavy chain. Expression level for AID or Iga was estimated by densitometry and was presented as a ratio to the respective loading control.
TNF-a upregulates AID expression via NF-kB signaling Z Duan et al. in cancer cells, we first treated NPC cells with different concentrations of TNF-a, then analyzed the expression level of AID. Results showed that TNF-a can upregulate AID expression in a dose-dependent manner ( Figure 3A) and also increases the expression level of AID mRNA ( Figure 3B). When an antibody was used to block TNF-a specifically, AID expression was decreased ( Figure 3C). These results suggested that TNF-a might regulate AID expression by activating transcription. The NF-kB-binding site in region 1 of the AID promoter is suggested to be responsive to viral infection and TNF-a signaling. 21 Therefore, we next examined whether NF-kB signaling is involved in TNF-a-induced AID expression. We used Bay 11-7082, an inhibitor of NF-kB signaling, and found that AID expression was downregulated when phosphorylation of IkBa and p65 were both decrease ( Figure 3D). These data provide evidence showing that TNF-a plays a role in AID expression mediated through NF-kB signaling in cancer cells.

AID expression is associated with CSR in cancer
It has been reported that AID-induced DNA lesions are achieved in part by the recruitment of the AID-activating kinase, PKA, exclusively to S regions during CSR. The activity of AID is also modulated by phosphorylation. Phosphorylation of serine 38 (Ser38) by the cAMP-dependent kinase PKA, is thought to be important for regulating AID activity. Here we confirmed that AID and PKA co-localize in the nucleus of cancer cells ( Figure 4A). Moreover, ChIP assays indicated that both AID and PKA target the Sa region of the Iga heavy chain locus ( Figure 4B). To clarify whether AID expression is involved in CSR of Iga in cancer, we transfected NPC cells with an AID-GFP overexpression plasmid, and found that the expression of the Iga heavy chain was increased when AID was overexpressed ( Figure 4C and D). Further study demonstrated that AID binds to the Sa region more efficiently when transfected with the AID-GFP plasmid ( Figure 4E and F). Finally, knocking down the expression of AID by shRNA transfection resulted in downregulation of the Iga heavy chain ( Figure 4G and H). These results suggested an association between AID expression and Iga class switching in cancer.

DISCUSSION
CSR is an essential process for expression of different Ig isotypes. In this study, we found that the Ig genes in cancer cells had completed CSR processing. AID is an inducible gene that plays a critical role in Ig CSR in B cells. Our research results indicated that AID is unexpectedly expressed in multiple cancer cell lines. Further studies demonstrated that TNF-a could induce AID expression through the NF-kB signaling pathway in NPC cells. Moreover, we verified that AID interacts with PKA and is involved in CSR of Ig genes in cancer.
EBV encoded LMP1 can upregulate AID expression in BL cells by mimicking CD40 signaling. Our previous studies demonstrated that the NF-kB signaling pathway is more active in LMP1-positive cancer cells. 7 However, when stimulated by TNF-a, we did not observe more abundant AID expression in LMP1-positive NPC cells. Additionally, the expression pattern of AID is not different between EBV-negative and EBV-positive gastric cancer cells (data not shown). Besides the stimulation of TNF-a, IL-4 might also play a role in the regulation of AID expression. We also found that in both CNE1 and CM cells, AID expression could be enhanced by IL-4 (data not shown). Thus, multiple cytokines could be involved in abnormal AID expression in cancer.
In addition to the transcriptional regulatory mechanisms of AID expression, AID is regulated posttranscriptionally. The microRNA-155 (miR-155) directly regulates AID expression by targeting the conserved site in the untranslated region. 30,31 The miR-181 family members are also differentially modulated during CSR and affect AID production. 32 These findings indicate that microRNAs play a role in AID expression in B lymphocytes and AID activity is also modulated by phosphorylation. Above all, phosphorylation of Ser38 by the cAMPdependent kinase PKA, is thought to be important for regulating AID activity. 33 A Ser38 to alanine mutant of AID showed diminished SHM and CSR activity on DNA targets. [34][35][36] CSR induced by AID targets tandemly repeated sequences of S regions throughout the Ig genes. Targeting of this motif is further enhanced by AID interaction with RPA, an ssDNA-binding protein involved in replication, recombination, and repair. 37 The AID-RPA interaction is dependent on the phosphorylation of Ser38 of AID at switch regions, a modification that might occur preferentially in B cells and that is reported to be required for CSR. [38][39][40]

CONFLICTS OF INTERESTS
The authors declare no financial or commercial conflict of interest.