Edyta Biskup

miR-122 Regulates p53/Akt Signalling and the Chemotherapy-Induced Apoptosis in Cutaneous T-Cell Lymphoma

Advanced cutaneous T-cell lymphoma (CTCL) is resistant to chemotherapy and presents a major area of medical need. In view of the known role of microRNAs (miRNAs) in the regulation of cellular signalling, we aimed to identify the functionally important miRNA species, which regulate apoptosis in CTCL. Using a recently established model in which apoptosis of CTCL cell lines is induced by Notch-1 inhibition by γ-secretase inhibitors (GSIs), we found that miR-122 was significantly increased in the apoptotic cells. miR-122 up-regulation was not specific for GSI-1 but was also seen during apoptosis induced by chemotherapies including doxorubicin and proteasome blockers (bortezomib, MG132). miR-122 was not expressed in quiescent T-cells, but was detectable in CTCL: in lesional skin in mycosis fungoides and in Sézary cells purified from peripheral blood. In situ hybridization results showed that miR-122 was expressed in the malignant T-cell infiltrate and increased in the advanced stage mycosis fungoides. Surprisingly, miR-122 overexpression decreased the sensitivity to the chemotherapy-induced apoptosis via a signaling circuit involving the activation of Akt and inhibition of p53. We have also shown that induction of miR-122 occurred via p53 and that p53 post-transcriptionally up-regulated miR-122. miR-122 is thus an amplifier of the antiapoptotic Akt/p53 circuit and it is conceivable that a pharmacological intervention in this pathway may provide basis for novel therapies for CTCL.

Notch1 as a potential therapeutic target in cutaneous T-cell lymphoma

Deregulation of Notch signaling has been linked to the development of T-cell leukemias and several solid malignancies. Yet, it is unknown whether Notch signaling is involved in the pathogenesis of mycosis fungoides and Sézary syndrome, the most common subtypes of cutaneous T-cell lymphoma. By immunohistochemistry of 40 biopsies taken from skin lesions of mycosis fungoides and Sézary syndrome, we demonstrated prominent expression of Notch1 on tumor cells, especially in the more advanced stages. The γ-secretase inhibitor I blocked Notch signaling and potently induced apoptosis in cell lines derived from mycosis fungoides (MyLa) and Sézary syndrome (SeAx, HuT-78) and in primary leukemic Sézary cells. Specific down-regulation of Notch1 (but not Notch2 and Notch3) by siRNA induced apoptosis in SeAx. The mechanism of apoptosis involved the inhibition of nuclear factor-κB, which is the most important prosurvival pathway in cutaneous T-cell lymphoma. Our data show that Notch is present in cutaneous T-cell lymphoma and that its inhibition may provide a new way to treat cutaneous T-cell lymphoma.

cMyc/miR-125b-5p Signalling Determines Sensitivity to Bortezomib in Preclinical Model of Cutaneous T-Cell Lymphomas

Successful/effective cancer therapy in low grade lymphoma is often hampered by cell resistance to anti-neoplastic agents. The crucial mechanisms responsible for this phenomenon are poorly understood. Overcoming resistance of tumor cells to anticancer agents, such as proteasome inhibitors, could improve their clinical efficacy. Using cutaneous T-cell lymphoma (CTCL) as a model of the chemotherapy-resistant peripheral lymphoid malignancy, we demonstrated that resistance to proteasome inhibition involved a signaling between the oncogene cMyc and miR-125b-5p. Bortezomib repressed cMyc and simultaneously induced miR-125b-5p that exerted a cytoprotective effect through the downmodulation of MAD4. Overexpression of cMyc repressed miR-125b-5p transcription and sensitized lymphoma cells to bortezomib. The central role of miR-125b-5p was further confirmed in a mouse model of T-cell lymphoma, where xenotransplantation of human CTCL cells overexpressing miR-125b-5p resulted in enhanced tumor growth and a shorter median survival. Our findings describe a novel mechanism through which miR-125b-5p not only regulates tumor growth in vivo, but also increases cellular resistance to proteasome inhibitors via modulation of MAD4.

MDM2 Inhibitor Nutlin-3a Induces Apoptosis and Senescence in Cutaneous T-Cell Lymphoma: Role of p53

P53 is rarely mutated in cutaneous T-cell lymphoma (CTCL) and is therefore a promising target for innovative therapeutic approaches. Nutlin-3a is an inhibitor of MDM2 (human homolog of murine double minute 2), which disrupts its interaction with p53, leading to the stabilization and activation of p53. To investigate the potential therapeutic use of nutlin-3a in CTCL, we screened CTCL lines Hut-78, SeAx, MyLa2000, Mac1, and Mac2a by measuring p53 levels after nutlin-3a treatment. In MyLa2000, Mac1, and Mac2a, we observed the increase in p53, indicating the fully functional p53. In the remaining cell lines, P53 mutation analysis identified a homozygous nonsense mutation (R196Stop in Hut-78) and a homozygous missense mutation (G245S in SeAx). In MyLa2000, Mac1, and Mac2a carrying wild-type P53, nutlin-3a induced apoptosis and senescence demonstrated by permanent G0/G1 cell-cycle block and expression of the senescence-associated β-galactosidase. This effect was abolished in cells in which p53 was silenced by small interfering RNA. Sézary cells lack functional p53 and were resistant to nutlin-3a. However, nutlin-3a potentiated the efficacy of conventional chemotherapeutics not only in cells with intact p53 but also in Hut-78, SeAx, and Sézary cells. Thus, targeting p53 by nutlin-3a may constitute a therapeutic approach in CTCL because of increased apoptosis and senescence of tumor cells.

miR-125b induces cellular senescence in malignant melanoma

BackgroundMicro RNAs (miRs) have emerged as key regulators during oncogenesis. They have been found to regulate cell proliferation, differentiation, and apoptosis. Mir-125b has been identified as an oncomir in various forms of tumours, but we have previously proposed that miR-125b is a suppressor of lymph node metastasis in cutaneous malignant melanoma. Our goal was therefore to further examine this theory.MethodsWe used in-situ-hybridization to visualise miR-125b expression in primary tumours and in lymph node metastasis. Then using a miRVector plasmid containing a miR-125b-1 insert we transfected melanoma cell line Mel-Juso and then investigated the effect of the presence of a stable overexpression of miR-125b on growth by western blotting, flow cytometry and β-galactosidase staining. The tumourogenicity of the transfected cells was tested using a murine model and the tumours were further examined with in-situ-hybridization.ResultsIn primary human tumours and in lymph node metastases increased expression of miR-125b was found in single, large tumour cells with abundant cytoplasm. A stable overexpression of miR-125b in human melanoma cell line Mel-Juso resulted in a G0/G1 cell cycle block and emergence of large cells expressing senescence markers: senescence-associated beta-galactosidase, p21, p27 and p53. Mel-Juso cells overexpressing miR-125b were tumourigenic in mice, but the tumours exhibited higher level of cell senescence and decreased expression of proliferation markers, cyclin D1 and Ki67 than the control tumours.ConclusionsOur results confirm the theory that miR-125b functions as a tumour supressor in cutaneous malignant melanoma by regulating cellular senescence, which is one of the central mechanisms protecting against the development and progression of malignant melanoma.

Notch signalling in primary cutaneous CD30+ lymphoproliferative disorders: a new therapeutic approach?

Background  The oncogenic potential of deregulated Notch signalling has been described in several haematopoietic malignancies. We have previously reported an increased expression of Notch1 in primary cutaneous CD30+ lymphoproliferative disorders, lymphomatoid papulosis and primary cutaneous anaplastic large‐cell lymphoma (pcALCL).

Proteasome inhibition as a novel mechanism of the proapoptotic activity of γ‐secretase inhibitor I in cutaneous T‐cell lymphoma

Background  We have previously discovered that Notch1 is expressed on malignant T cells in cutaneous T‐cell lymphoma (CTCL), and is required for survival of CTCL cell lines. Notch can be inhibited by γ‐secretase inhibitors (GSIs), which differ widely in their ability to induce apoptosis in CTCL.

The importance of Notch signaling in peripheral T-cell lymphomas

Abstract Peripheral T-cell lymphomas (PTLs) represent an area of high medical need. Previously, we demonstrated high expression of Notch, a known oncogene, in primary cutaneous anaplastic large cell lymphoma (ALCL). In this study, we performed immunohistochemical staining for Notch1 in lymph nodes from PTL not otherwise specified (PTL-NOS) and systemic ALCL (ALK+ and ALK−) and report a similar distribution among the three subgroups: Negative, moderate and strong expression was, respectively, 18%, 27% and 55% for PTL-NOS (33 cases), 20%, 0% and 80% for ALCL ALK+ (10 cases) and 45%, 22% and 33% for ALCL ALK− (nine cases) (p > 0.05). In the ALK+ ALCL cell line, Karpas-299, pharmacological inhibition of Notch with γ-secretase inhibitor (GSI) I was far more potent than with GSI IX, XX and XXI with regard to cell viability and apoptosis. In conclusion, PTL tumor cells have prominent Notch1 expression and treatment with Notch inhibitors has cytotoxic effects.

No donor age effect of human serum on collagen synthesis signaling and cell proliferation of human tendon fibroblasts.

The aging process of tendon tissue is associated with decreased collagen content and increased risk for injuries. An essential factor in tendon physiology is transforming growth factor-β1 (TGF-β1), which is presumed to be reduced systemically with advanced age. The aim of this study was to investigate whether human serum from elderly donors would have an inhibiting effect on the expression of collagen and collagen-related genes as well as on cell proliferative capacity in tendon cells from young individuals. There was no difference in systemic TGF-β1 levels in serum obtained from young and elderly donors, and we found no difference in collagen expression when cells were subjected to human serum from elderly versus young donors. In addition, tendon cell proliferation was similar when culture medium was supplemented with serum of different donor age. These findings suggest that factors such as the cell intrinsic capacity or the tissue-specific environment rather than systemic circulating factors are important for functional capacity throughout life in human tendon cells.

Ellipticine induces apoptosis in T-cell lymphoma via oxidative DNA damage

Abstract The tumor suppressor p53 is often mutated in human cancers. Restoring its antitumor activity has been shown to be a promising therapeutic approach for cancer treatment. Here we analyzed the activity and mechanism of a p53 reactivator, ellipticine, in a cellular model of cutaneous T-cell lymphoma (CTCL), a disease that is progressive, chemoresistant and refractory to treatment. We tested the effect of ellipticine in three cell lines with different p53 status: MyLa2000 (p53wt/wt), SeAx (G245Sp53) and Hut-78 (R196Stopp53). Ellipticine caused apoptosis in MyLa2000 and SeAx and restored the transcriptional activity of G245Sp53 in SeAx. However, p53 siRNA knockdown experiments revealed that p53 was not required for ellipticine-induced apoptosis in CTCL. The lipophilic antioxidant α-tocopherol inhibited ellipticine-dependent apoptosis and we linked the apoptotic response to the oxidative DNA damage. Our results provide evidence that ellipticine-induced apoptosis is exerted through DNA damage and does not require p53 activation in T-cell lymphoma.