POTENTIAL ROLE OF LIN28/LET-7 AXIS IN SEZARY SYNDROME

Sézary Syndrome (SS) is a rare leukemic cutaneous T-cell lymphoma (CTCL) with an aggressive clinical course and a survival of 1-5 years. It is characterized by erythroderma, generalized lymphadenopathy and the presence of neoplastic CD4+ lymphocytes (Sézary cells) in the skin, lymph nodes and peripheral blood. It is well known that deregulation in STAT3 signaling is one of the molecular mechanism involved in CTCL pathogenesis and cancer progression and many studies demonstrated the efficacy of JAK/STAT inhibitors to induce apoptosis in Sézary cells. However the cause of SS is still unknown and until now, no specific therapy is available for this disease. Recently, microRNA (miRNA) profiling analysis in SS revealed a possible role of miRNAs in the pathogenesis of the disease and identified specific miRNAs as possible new targets in SS treatment. Particularly miR-21, miR-486 and miR-214 were found upregulated in SS while let-7 family members were found downregulated. miRNAs are small non-coding RNAs that regulate gene expression via post transcriptional silencing of target genes. They have important roles in fundamental biological processes such as cell development, differentiation, proliferation and apoptosis. Let-7 family members are widely viewed as tumor suppressor miRNAs and they are strongly downregulated in many cancers. Particularly let-7a is known to target many oncogenes including c-Myc and its upregulation inhibits cell proliferation by reducing the expression of c-Myc. Another target of let-7a is c-MET and its downstream signaling pathways such as RAS, PI3K, STAT3 and β-catenin pathways. Let-7b has several oncogenes as targets such as HMGA1, HMGA2, CCND2 and KRAS, and loss of expression of let-7b has been demonstrated in many cancers comprising acute lymphoblastic leukemia. Many studies demonstrated that downregulation of let-7 is due to its direct interaction with LIN28A or LIN28B. LIN28 depletion resulted in specific increases in all let-7 family members. On contrast Nishi et al demonstrated that let-7b is downregulated in leukemic cells via DNA hypermethylation of its regulatory region. Moreover in pancreatic cancer cell lines let-7 downregulation has been reported to be linked to a constitutively-active STAT3. In two independent studies (Narducci et al. and Corti et al.) a downregulation of some members of let-7 family, particularly let-7a and let-7b, was demonstrated in SS by using microRNA microarrays. Particularly, in a previous unpublished study from our group we found a clear split in miRNA expression between SS patients and controls. Of particular interest let-7b was the most downregulated in our patients series and comparison of miRNA profile and CGH data showed that all patients with gains in 8q (MYC) have also let-7b downregulation. We found 24 miRNA differentially expressed between SS patients and healthy controls, including miR-214, miR-486 and miR-21 yet described and validated by Narducci et al. and Qin et al. Expression of let-7 members is controlled by MYC binding to their promoters and levels of let-7 have been reported to decrease in models of MYC-mediated tumorigenesis. Amplification of 8q (MYC) found on the same patients with let-7b downregulation and LIN28/let-7b link with STAT3/miR21 activation suggest a possible key role of let-7 family members (particularly let-7b) in SS. Finally, let-7 family members expression lead to apoptosis by Bcl-XL repression. In a recent study, Adams et al demonstrates that, in a wide group of haematologic malignancies, Let-7b downregulation lead to apoptosis blockage and that restoring let-7 normal expression downregulates Bcl-XL with a consequent upregulation of caspase-3 and apoptotic activity in neoplastic cells. 26 patients affected by SS and 15 healthy donors were recruited at Dermatology Department of Fondazione IRCCS Ca’ Granda – Ospedale Maggiore Policlinico of Milan. Each group (patients and controls) released their informed consent according to the Declaration of Helsinki Principles. Heparinized (or EDTA) blood were drawn from patients and controls, and mononuclear cells were isolated by Ficoll density centrifugation. Subsequently, CD4+ T-cells were isolated by using magnetic-beads conjugated antibodies (CD14 negative selection and CD4 positive selection). Depending on material availability CD4+ lymphocytes from patients and HD were used for RNA and/or protein extraction. Total RNA samples will be converted into complementary DNA (cDNA) and used to asses expression levels of let-7b, let-7c, Lin28A, Lin28B, c-Myc, CCND1, HMGA1 and HMGA2 by quantitative reverse transcriptase PCR (qRT-PCR). The expression levels were standardized using RNA18S gene as housekeeping. Apoptosis molecules were evaluated by means of protein array experiment. Protein array is a glass slide based array in which is possible to investigate expression levels of different proteins at the same time. The goal was to demonstrate whether let-7s downregulation could be related to a diminished apoptosis activity in SS. This hypothesis would allow targeting let-7s to restore apoptosis in neoplastic cells. By q-RT PCR both let-7 miRNAs (let-7b and let-7c) showed differential expression with greater downregulation in SS than in healthy donors. Let-7b resulted downregulated in 8/18 (44%) patients and in 0/9 (0%) controls while let-7c was shown downregulated in 7/18 (39%) patients and 0/9 (0%) HD. 2 patients showed a downregulation of both let-7b and let-7c miRNAs (11%). Globally, 13/18 (72%) patients had a downregulation in at least one let-7. Analyzing expression levels of Lin28A and Lin28B both resulted unexpressed in HD. In SS patients Lin28B was found unexpressed while Lin28A was found only in a 7 out 18 SS patients. However, 5 out of 7 patients with Lin28A overexpression had a let-7c downregulation. Let-7c downregulation could be considered a consequence of direct repression by Lin28A. Moreover, in order to investigate whether let-7 expression influenced response to therapy all SS patients were divided in two groups. “responders” and “non responders” to therapy. Let-7 downregulation seemed to be linked to response to therapy. In fact, number of “non responder” patients in let-7 downregulated group was much higher than those in let-7 normal expressed group. Among the 43 protein studied by means of apoptosis array 11 proteins showed a statistically significative difference between SS and HD. Among these pro-apoptotic factors bad, bax, bcl-w and BID and cyclin-dependent kinase inhibitors p21, p27 and p53 resulted less expressed in SS than in normal controls. Particularly, p53 showed very low levels in patients with let-7 downregulation. On the other hand inhibitors of apoptosis family members cIAP-2 and XIAP and heat shock protein 60 (HSP60) showed a higher expression in SS than in HD. qRT-PCR experiments confirmed let-7b and let-7c downregulation in more than 70% of SS patients versus 0% of HD. In the present study a significant linear correlation was found between Lin28A expression and let-7c downregulation. This suggest that let-7c downregulation in SS is due to a Lin28A reactivation. Finally, about apoptosis pathways, some apoptosis related molecules were found differentially expressed in SS than in HD. Although no evidences were found for a direct role of let-7s in regulating apoptosis in SS, some de-regulated proteins could collaborate to chemoresistance in SS. Taken together these data suggest the existence of complex mechanisms regulating apoptosis and drug resistance in SS. Further studies, including functional studies, are needed to better understand the role of each molecule in this complex network.