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Li H.,Karolinska Institutet | Lakshmikanth T.,Karolinska Institutet | Garofalo C.,University of Catanzaro | Enge M.,Karolinska Institutet | And 7 more authors.
Cell Cycle | Year: 2011

Escape of tumor cells from cell-intrinsic barrier mediated by tumor suppressors and cell-extrinsic barrier mediated by the immune system is crucial for tumorigenesis. Growing evidence suggests that reactivation of tumor suppressor function or restoration of anticancer immunity are promising strategies for anticancer therapy due to their high potential to combat cancer. p53, a key tumor suppressor, represses tumorigenesis by eliciting growth arrest, apoptosis or senescence in cancer cells. Here, we unravel that, apart from these cell-autonomous effects, p53 activates the innate immune response against cancer cells. Our results show that pharmacological reactivation of p53 can stimulate the expression of ULPB2, a ligand for NK cell activating receptor NKG2D in human tumor cells of different origin, which enhance the susceptibility of tumor cells to NK cell-mediated killing. The molecular mechanism controlling ULPB2 expression by p53 is neither ATM/ ATR- nor caspase-dependent. Using several approaches, we identified p53 as a direct transcriptional regulator of ULBP2 and found a p53 response element within ULBP2 gene, which confers the p53 regulation. Furthermore, we demonstrated that demethylation of p53-binding region within ULBP2 gene was required for p53-dependent induction of ULPB2, which can be achieved via repression of DNA methyltransferases (DNMTs) by p53. This molecular evidence for the direct control of immunosurveillance by p53 links tumor suppressor activation to innate immune stimuli and provides a possibility to integrate cell-extrinsic and -intrinsic defenses against tumorigenesis by pharmacological activation of p53, which may increase the probability to achieve a durable therapeutic success. © 2011 Landes Bioscience. Source

Degl'Innocenti D.,Molecular Mechasnisms Unit | Romeo P.,Molecular Mechasnisms Unit | Tarantino E.,Fondazione Istituto Nazionale Dei Tumori | Sensi M.,Human Tumors Immunobiology Unit | And 9 more authors.
Endocrine-Related Cancer | Year: 2013

Thyroid carcinomas derived from follicular cells comprise papillary thyroid carcinoma (PTC), follicular thyroid carcinoma, poorly differentiated thyroid carcinoma (PDTC) and undifferentiated anaplastic thyroid carcinoma (ATC). PTC, the most frequent thyroid carcinoma histotype, is associated with gene rearrangements that generate RET/PTC and TRK oncogenes and with BRAF-V600E and RAS gene mutations. These last two genetic lesions are also present in a fraction of PDTCs. The ERK1/2 pathway, downstream of the known oncogenes activated in PTC, has a central role in thyroid carcinogenesis. In this study, we demonstrate that the BRAF-V600E, RET/PTC, and TRK oncogenes upregulate the ERK1/2 pathway's attenuator cytoplasmic dual-phase phosphatase DUSP6/MKP3 in thyroid cells. We also show DUSP6 overexpression at the mRNA and protein levels in all the analysed PTC cell lines. Furthermore, DUSP6 mRNA was significantly higher in PTC and PDTC in comparison with normal thyroid tissues both in expression profile datasets and in patients' surgical samples analysed by real-time RT-PCR. Immunohistochemical and western blot analyses showed that DUSP6 was also overexpressed at the protein level in most PTC and PDTC surgical samples tested, but not in ATC, and revealed a positive correlation trend with ERK1/2 pathway activation. Finally, DUSP6 silencing reduced the neoplastic properties of four PTC cell lines, thus suggesting that DUSP6 may have a pro-tumorigenic role in thyroid carcinogenesis. © 2013 Society for Endocrinology. Source

Del Vecchio M.,Unit of Medical Oncology 2 | Mortarini R.,Human Tumors Immunobiology Unit | Canova S.,Unit of Medical Oncology 2 | Di Guardo L.,Unit of Medical Oncology 2 | And 9 more authors.
Clinical Cancer Research | Year: 2010

Purpose: To assess the clinical and biological activity of the association of bevacizumab and fotemustine as first-line treatment in advanced melanoma patients. Experimental Design: Previously untreated, metastatic melanoma patients (n = 20) received bevacizumab (at 15 mg/kg every 3 weeks) and fotemustine (100 mg/m2 by intravenous administration on days 1, 8, and 15, repeated after 4 weeks) in a multicenter, single-arm, open-label, phase II study. Primary endpoint was the best overall response rate; other endpoints were toxicity, time to progression (TTP), and overall survival (OS). Serum cytokines, angiogenesis, and lymphangiogenesis factors were monitored by multiplex arrays and by in vitro angiogenesis assays. Effects of fotemustine on melanoma cells, in vitro, on vascular endothelial growth factor (VEGF)-C release and apoptosis were assessed by ELISA and flow cytometry, respectively. Results: One complete response, 2 partial responses (PR), and 10 patients with stable disease were observed. TTP and OS were 8.3 and 20.5 months, respectively. Fourteen patients experienced adverse events of toxicity grade 3-4. Serum VEGF-A levels in evaluated patients (n = 15) and overall serum proangiogenic activity were significantly inhibited. A significant reduction in VEGF-C levels was found in several post-versus pretherapy serum samples. In vitro, fotemustine inhibited VEGF-C release by melanoma cells without inducing significant cell death. Serum levels of interleukin (IL)-10 and IL-12p70 showed the highest levels in sera of PR patients, compared with patients with stable or progressive disease whereas IL-23 showed the opposite pattern. Conclusions: The combination of bevacizumab plus fotemustine has clinical activity in advanced melanoma and promotes systemic modulation of angiogenesis and lymphangiogenesis factors. ©2010 AACR. Source

Anichini A.,Human Tumors Immunobiology Unit | Molla A.,Human Tumors Immunobiology Unit | Vegetti C.,Human Tumors Immunobiology Unit | Bersani I.,Human Tumors Immunobiology Unit | And 9 more authors.
Cancer Research | Year: 2010

CD8+ T cells at the earliest stage of effector generation have not been identified at tumor site of melanoma patients. Such early effectors, if present, should be characterized by a specific phenotype, distinct from that expressed at later stages of the antigen-induced differentiation program, by short-lived effector cells, memory precursors, and terminal effectors. Here, we show that neoplastic tissues from primary and metastatic lesions of melanoma patients contain a subset of CD8+ T cells expressing FOXP3. CD8 + FOXP3+ CD25+ T lymphocytes were found in tumor-invaded lymph nodes (TILN), s.c. metastases, and advanced primary lesions. Their frequency was significantly higher in TILN compared with tumor-free lymph nodes or with peripheral blood and in primary tumors compared with TILN. CD8+ FOXP3+ T cells did not express markers of regulatory [CTLA-4, CCL4, interleukin-10 (IL-10), transforming growth factor-β1], exhausted (PD-1), or senescent (CD57) CD8+ T lymphocytes. Instead, this subset showed an antigen-experienced "EM1" phenotype (CCR7 -. CD45RA- CD28+ CD27+) and exhibited a CD127-, KLRG1-, HLA-DR+, CD38 +, T-bet+, perforin+ "early effector" profile predicted by current models. CD8+ FOXP3+ T cells produced IFN-γ on short in vitro activation, recognized autologous tumor by CD107a mobilization, and expressed Ki-67 on ex vivo analysis. In response to autologous tumor plus IL-2/IL-15, the CD8+ FOXP3+ T cells proliferated promptly and showed competence for differentiation (downregulation of CD27 and upregulation of T-bet). These results suggest development of early phases of antitumor immunity even in advanced melanoma. Moreover, the CD8 + FOXP3+ "early effector" subset may be an invaluable tool for monitoring immunity at tumor site. ©2010 AACR. Source

Loria R.,Regina Elena Cancer Institute | Bon G.,Regina Elena Cancer Institute | Perotti V.,Human Tumors Immunobiology Unit | Gallo E.,Regina Elena Cancer Institute | And 10 more authors.
Oncotarget | Year: 2015

We used whole genome microarray analysis to identify potential candidate genes with differential expression in BRAFV600E vs NRASQ61R melanoma cells. We selected, for comparison, a peculiar model based on melanoma clones, isolated from a single tumor characterized by mutually exclusive expression of BRAFV600E and NRASQ61R in different cells. This effort led us to identify two genes, SEMA6A and MICAL1, highly expressed in BRAF-mutant vs NRAS-mutant clones. Real-time PCR, Western blot and immunohistochemistry confirmed preferential expression of Sema6A and Mical1 in BRAFV600E melanoma. Sema6A is a member of the semaphorin family, and it complexes with the plexins to regulate actin cytoskeleton, motility and cell proliferation. Silencing of Sema6A in BRAF-mutant cells caused cytoskeletal remodeling, and loss of stress fibers, that in turn induced cell death. Furthermore, Sema6A depletion caused loss of anchorage-independent growth, inhibition of chemotaxis and invasion. Forced Sema6A overexpression, in NRASQ61R clones, induced anchorage-independent growth, and a significant increase of invasiveness. Mical1, that links Sema/PlexinA signaling, is also a negative regulator of apoptosis. Indeed, Mical-1 depletion in BRAF mutant cells restored MST-1-dependent NDR phosphorylation and promoted a rapid and massive NDR-dependent apoptosis. Overall, our data suggest that Sema6A and Mical1 may represent new potential therapeutic targets in BRAFV600E melanoma. Source

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