The Mechanism Of Ig Gene Class Switch Recombination And Ig Alpha Heavy Chain Expression In Cancer Cells

It is traditionally believed that the production of immunoglobulin (Ig) molecules is restricted to B lymphocytes. All cells in human bodies include germline Ig genes, when stimulated by foreign antigens and affected by various regulators, the Ig genes undergo V(D)J recombination, class switch recombination (CSR) and somatic hypermutation (SHM), only then can immunoglobulins be expressed in B lymphocytes.We previously found the transforming Tx gene, which was cloned from the gDNA library of nasopharyngeal carcinoma cell line CNE2, is an abnormal Ig κ gene lacked variable regions. This innovative discovery suggested the possibility of Ig expression in cancer cells. Other research groups have subsequently reported Ig expression and secretion in non-lymphoid cancer cells. Scientists have demonstrated the biological functions and the molecular mechanisms of Ig expression in cancers, including the basis of V(D)J recombination. However, whether the Ig genes undergo class switch and the mechanisms underlying how cancer cells initiate CSR are still undetermined.To confirm whether the Ig genes experience class switch recombination in cancer cells, we first analyzed the products of CSR in several cancer cell lines. Results showed there are both Ig a heavy chain and its transcript Ig VDJ-Ca expression in cancer cells. Furthermore, we have detected the hallmarkers of CSR in genomic and transcriptional level. CSR will generate integrated Ig a gene VDJ-Ca and looped-out circle Sa-Sμ., by amplifying the connectors of these two hallmarkers, we found that integrated Ig a gene VDJ-Ca and looped-out circle Sa-Sp, are present in cancer cells. These results indicated that Ig genes in cancer cells have completed CSR in genomic level.To illuminate the molecular mechanism of class switch, we focus on two crucial factors associated with CSR in B lymphocytes, including activation-induced cytidine deaminase (AID) and Ig Iα-Cα germline transcript. We first demonstrated that AID is abnormally expressed in nasopharyngeal carcinoma (NPC) tissues and several cancer cell lines. Further studies indicated that TNF-a may play an important role in AID expression via NF-κB signaling. Moreover, in order to verify if AID participate in CSR in cancer cells, we found that AID can interact with PKA by immunofluorescence-confocal assays. We also confirmed that AID and PKA can target to Sa region of Ig genes by ChIP assays, which is essential for Ig a expression.To explore the molecular basis of Ig Iα-Cα germline transcription, we focus on the Ig la promoter and transcription factor which is essential for Ig la promoter activation. We first constructed a report plasmid including Ig la promoter and determined whether the Ig la promoter is activated in cancers. Results showed that the Ig la promoter was highly activated in NPC cells. Through bioinformatic analysis, we found several binding sites of various transcription factors, including NF-κB and PU.1, in the Ig Iα promoter. Further studies confirmed that an ETS family member, Ets-1, can bind to the PU.1motif and then transactivate the Ig Iα promoter. The activity of the Ig la promoter was decreased by knockdown of Ets-1. Also, Ig Iα-Cα germline transcription and Ig α expression were attenuated after knockdown of Ets-1by specific small interfering RNA (siRNA). Furthermore, stimulation of TGF-β1can upregulate the activity of the Ig la promoter, and the expression of Ets-1and Iga heavy chain in cancer cells was dose-dependently increased by TGF-β1. These results indicate that activation of the Ig la promoter by transcription factor Ets-1is a critical pathway and provide a novel mechanism for Ig a expression in non-B lymphocyte cancers.From this study, we confirmed that stimulation of TNF-a can upregulate AID expression via NF-κB signaling, and TGF-β1can increase Ig Ia-Ca germline transcription by upregulating Ets-1. These two discoveries indicated the molecular mechanism of class switch recombination, which is curial for Ig a expression in cancer cells. These results will provide some hints for the biological functions of cancer-derived Igs and cancer development.