TCL1A interacts with TP63 and enhances the survival of Raji Burkitt lymphoma cell line (2017)

TCL1A was initially discovered almost three-two decades ago and is considered an important oncogene (Russo et al, 1989). TCL1A protein is normally expressed in fetal tissues and healthy lymphocytes, and when overexpressed it causes leukaemias and lymphomas (Madani et al, 1996). TCL1A expression in mouse models induces the development of a leukaemia that is very similar to the human CLL (chronic lymphocytic leukaemia) (Bichi et al, 2002). TCL1A is a co-activator of AKT and exerts its role by favouring AKT phosphorylation (Laine et al, 2000). More recently, we isolated TCL1A-interacting proteins by using a proteomic approach based on mass spectrometry. Among the most interesting TCL1A partners, we studied ATM (Ataxia-telangiectasia mutated) (Gaudio et al, 2012) and HSP70 (Heat shock protein 70) (Gaudio et al, 2013). The physical interaction between TCL1A and ATM leads to a stronger NF-κB activation, which comprises the phosphorylation and degradation of IκB-alpha and the translocation of RELA into the nucleous (Gaudio et al, 2012). HSP70 interacts with TCL1A and protect it from degradation (Gaudio et al, 2013). Based on its central role in the PI3K/AKT and NF-κB pathways, translation of TCL1A inhibition into the clinics is therefore highly desirable. There are currently no inhibitors of TCL1A, although peptides isolated from the TCL1A protein sequence show potential activity. A TCL1A peptide called AKT-in (Hiromura et al, 2004), spanning a region of TCL1A, can inhibit AKT, also confirming the crucial role of TCL1A as an activator of AKT. TCL1A peptides for immunotherapy have been recently patented (patent: WO 2013075105 A3): they bind to major histocompatibility complex (MHC) I (HLA-A2) on tumour cells or other antigen-presenting cells and are recognized by T-cell receptors on T cells. These peptides may be therapeutically used to treat leukaemia and lymphoma (Weng et al, 2012). Here, we demonstrated that TCL1A interacts with the TP63 protein. Moreover, the recognition of the AP1 consensus (ATGAGTCaT) (nt 4113 – nt 4121) (Humphries et al, 2013) on the TP63 promoter sequence by TCL1A enhances TP63 luciferase activity. We searched for other endogenous TCL1A interacting proteins in order to better clarify its role in lymphoma. To this purpose, protein lysates from Burkitt lymphoma cells (Raji cell line) were immunoprecipitated with anti-IgG (experimental control) and with anti-TCL1A antibody. TCL1A protein and its putative partners was isolated using a A/G agarose resin. Purified proteins were digested by trypsin, and protein constituents were identified by LC-MS/MS (Table 1). Among the identified candidate interactors (present only in the TCL1A immunoprecipitated sample), we focused on TP63, given its role of interest in lymphomas (Humphries et al, 2013). The physical interaction between endogenous TCL1A and TP63 proteins was investigated and confirmed in the Raji cell line by co-immunoprecipitation (Fig 1A). Endogenous proteins from Raji cells were immunoprecipitated with anti-TCL1A and solubilized and digested by trypsin. Protein constituents were identified by liquid chromatography tandem mass spectrometry (LC-MS/MS). Inspection of LC-MS/MS data was undertaken to assess exclusive presence of mass peaks belonging to candidate partner proteins in samples from cells immunoprecipitated with anti-TCL1A and not from cells immunoprecipitated with anti-IgG (control sample). These studies were performed as previously described (Gaudio et al, 2012, 2013). Trans-Activating TP63 is largely expressed in lymphomas, including Burkitt's lymphoma (Fukushima et al, 2006), where it behaves as an oncogene (Humphries et al, 2013). We confirmed the oncogenic role of TP63 by inducing its down-regulation in the Raji cell line (Fig 1B). Cells were transfected with 100 ng of small interfering RNA (siRNA) targeting TP63 and/or TCL1A. Interestingly, cells treated with siRNA anti-TCL1A showed down-regulation of phosphorylated NFKBIA (Ser 32) and up-regulation of total NFKBIA and RELA (Fig 1B); the combination of both siRNAs was able to reduce both AKT and phospho-AKT (Ser 473) protein levels (Fig 1B) and cell growth measured after 72 h (Fig 1C). To determine whether TCL1A expression directly enhances the transactivating function of TP63 we performed luciferase assays (Fig 1D). HEK293 cells were transfected with TP63-promoter-LUC vector with/without TCL1A protein (induced by adding TCL1A expression vector) and luciferase activity was measured after 24 h (Fig 1D). The transcriptional activity of the TP63 promoter sequence was effectively enhanced by TCL1A. A similar experiment was also conducted in Raji cells; Briefly, cells were transfected with TP63-promoter-LUC vector, TCL1A protein expression was reduced by using siRNA anti TCL1A and luciferase activity was measured after 24 h (Fig 1E). The transcriptional activity of the TP63 promoter sequence was effectively enhanced by the endogenous presence of TCL1A. This work proposes, for the first time, the physical interaction between TP63 and TCL1A protein and the concept that TCL1A modulates TP63 transcriptional activity by interacting with the AP1 sequence located on its promoter. Interestingly, our findings contribute to enlarge the array of potentially targetable proteins involved in both leukaemogenesis and lymphomagenesis in order to generate more efficient drugs for clinical use. This work is lovingly dedicated to the memory of Pietro Gaudio, who died on March 11, 1996. This study was possible thanks to a generous donation in loving memory of Carmelo Stillitani through the Fondazione Università Magna Græcia (FUMG) of Catanzaro. This Article is based upon work from the European Cooperation in Science and Technology (COST) Action CA15135, supported by COST. E.G. designed the experiments. S.P., F.P., S.D'A., A.N., E.G. performed research experiments. F.B., C.M.C., F.T., R.I.A. advising contribution. E.G., F.T. wrote the paper. All authors critically reviewed the manuscript and approved the final version. The authors declare no competing financial interest. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.