Study On The Role Of LIM Domain Protein FHL1C In T-cell Acute Lymphoblastic Leukemia Proliferation And Apoptosis

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive neoplasm resulted from immature T-cells. At current, although chemotherapy is still chosen as one main treatment strategies, more than 50% patients still have high risk of relapse; their mean of survival rates is less than 1 year. Even to carry out heterogeneity's hematopoietic stem cells transplantation, Event-free survival (EFS) rates are only 30%~50%. Therefore, it is very meaningful to improve the long term of T-ALL patients'survival by deeply exploring the molecular mechanism of T-ALL development, and looking for new targeted therapies.Notch/RBP-J signaling pathway is high conservation during evolution, and plays a pivotal role in the regulation of the progenitor as well as stem cell differentiation by mediating cell-cell interaction, and its abnormal activation related with many different tumors development. The first evidence of oncogenic Notch signaling was observed in T-ALL. A large amount of reports show the main molecular mechanism of T-ALL development and maintenance are attributed to a translocation of the Notch gene (coded in Chromosome 9) on to the T cell receptor (TCR) beta chain locus (located in chromosome 7) and Notch mutations. Although currently it is reported small moleculeγ-secretase inhibitors (GSIs), which block a critical proteolytic step required for Notch activation, can be applied for T-ALL treatment in phase I clinical trial, the clinical outcome of GSIs is not ideal. The main reasons are that on one handγ-secretase is not specific for Notch receptors, on another hand GSIs can only effect on ligand-dependent Notch activation, not on ligand-independent Notch activation resulted from genetic choromosome translocation and point mutations as mentioned above. Indeed, over 50% of T-ALL patients are indentified with activating Notch1 mutations. Thus, it is very imperative to look for more effective Notch signaling inhibitors for the treatment of T-ALL.DNA binding protien RBP-J, a key transcription factor of Notch signal, can mediate four types of Notch receptors transactivation in the nucleus. Aster et al. found Notch1 mutants can regulate downstream genes expression involved in T-ALL through interating with transcription factor RBP-J, a dominant-negative fragments of MAML1(DN-MAML1) had been shown to anatagonize Notch1 activation when expressed in T-ALL cell lines. Later, Brandner et al.designed one stable -helical peptides SAHM1 (Stapled -helical peptides derived from MAML1) based on DN-MAML1 structure, and then verify SAHM1 can directly impede the assembly of Notch transactivation complex in nucleus and finally reduce tumor cell proliferation and promote tumor cell apoptosis by suppressing the activating Notch signal. Thus, compared with GSIs, DN-MAML and SAHM1 can inhibit Notch activation more efficiency due to its direct inhibition of Notch signal on transcription factor. However, as multifunctional transcription activator, MAML1 is also not specific for Notch signaling. Regarding on this point, the treatment of T-ALL with DN-MAML1 and SAHM1 is still on the way. LIM domain protein KyoT2, one RBP-J interacting protein, can suppress RBP-J-mediated Notch transactivation by competing with NIC. With deep investigation, our group found KyoT2 might suppress RBP-J-mediated Notch transactivation by recruiting Polycomb suppression complex including RING1 and HPC2 through the LIM protein, and then verified KyoT2 also can repress Notch transactivatin by PIAS1-meidiated Sumoylation process. Mammalian FHL1C is homologous of murine KyoT2, and belongs to FHL1 family. Recently, many evidences show FHL1 is down-regulated in a variety of cancers by comparative microarray profiling and immunohistochemical analysis, such as in lung, breast, colon and brain cancers, and so on. Also we find the low expression level of FHL1C in T-ALL patients contrasting to normal people. In addition, we find KyoT2 knock-out mouse don't show any abnormal on its survival and physiological function. All tHese studies indicate that over-expressed FHL1C (KyoT2) in T-ALL might treat T-ALL by inhibiting Notch activation. According to this idea, we have carried out some experiments as follow.The main results:1. We successfully construct FHL1C eukaryotic expression vectors (pCMV-myc-FHL1C and pEGFP-C1-FHL1C) and lentiviral expression vector (pLenti/V5-FHL1C-IRES-GFP), and verify FHL1C can be expressed by cell transfection, Western-blot and FACS analysis.2. Using Amaxa nucleic transfection instrument, we can express FHL1C in T-ALL cell line (Jurkat cells) with high transfection efficiency. With it, we find over-expressed FHL1C can promote Jurkat cell apoptosis, but not influence Jurkat cell proliferation by cell number count, FACS analysis, Annexin/PI staining, Hoechst staining and transmission election microscopy.3. We primarily explore the molecular mechanism of FHL1C induced Jurkat cell apoptosis. We confirm FHL1C can suppress Notch transactivation as KyoT2 did by luciferase reporter assay, and find the expression of some downstream genes of Notch signaling , which are related with T-ALL, are down-regulated, such as Hes1,Hes5 and c-Myc; Some apoptosis genes, such as Fas and Caspase-3, are up-regulated; and the expression of some anti-apoptosis genes, such as Bcl-2 and Bcl-xl, are reduced.In a word, our study indicate that over-expression of FHL1C in T-ALL cell line can induce cell apoptosis. The possible mechanism is that FHL1C inhibit some downstream genes expression , such as Hes1,Hes5 and c-Myc that involved in T-ALL development, and at the same time promote some apoptosis related genes expression and reduce some anti-apoptosis genes expression. THese results suggest FHL1C can be developed as one target to treat T-ALL. THese studies are very helpful to elucidate the mechanism of FHL1C induced T-ALL cell apoptosis in future, and shed light on designing new Notch inhibitor based on FHL1C to treat T-ALL in the end.