Search

Supplementary online material. (DOCX 119Â kb)

Confronto della cartella sanitaria prevista per rischi convenzionali e rischi da radiazioni ionizzanti.

Studio statistico delle pubblicazioni su riviste peer review su temi di medicina del lavoro nell'ultimo decennio.

Context The resistance of tumors to antiangiogenic therapies is becoming increasingly relevant. There are currently no validated predictive biomarkers for selecting which cancer patients will benefit from antiangiogenic therapy. Also lacking are resistance biomarkers that can identify which escape pathways should be targeted after tumors develop resistance to VEGF treatment. Recent studies showed that anti-VEGF treatment can make tumor cells more aggressive and metastatic. However, the mechanisms and mediators of this are unidentified. Objective We aimed this study at directly identifying the tumor cell-initiated mechanisms responsible for the resistance of pancreatic cancer to anti-VEGF treatment. Methods We established and validated two murine models of human pancreatic cancer resistant to the VEGF-specific antibody bevacizumab in vivo. We used a genome-wide analysis to directly identify which tumor-secreted factors were overexpressed by pancreatic cancer cells that were resistant to anti-VEGF treatment. Results Rather than direct proangiogenic factors, we identified several proinflammatory factors that were expressed at higher levels in cells resistant to anti-VEGF treatment than in treatment-sensitive control cells. These proinflammatory factors acted in a paracrine manner to stimulate the recruitment of CD11b+ proangiogenic myeloid cells. Also, we found that secreted factors overexpressed by anti-VEGF treatment-resistant pancreatic cancer cells acted in an autocrine manner to induce epithelial-to-mesenchymal transition (EMT) and were thus responsible for increased aggressiveness of bevacizumab-resistant pancreatic tumors. Conclusions Our results identified proinflammatory factors and EMT markers as potential biomarkers for selecting patients with pancreatic cancer for antiangiogenic therapy., JOP. Journal of the Pancreas, Vol 14, N° 5S (2013): September (Suppl.) - p. 528-602

Context TAK1, a mitogen-activated protein kinase kinase kinase, functions in the activation of nuclear factor kappaB (NF-kappaB) and activator protein-1, which can suppress proapoptotic signaling pathways and thus promote resistance to chemotherapeutic drugs. However, it is not known if inhibition of TAK1 is effective in reducing chemoresistance to therapeutic drugs against pancreatic cancer. Methods: NF-kappaB activity was measured by luciferase reporter assay in human pancreatic cancer cell lines AsPc-1, PANC-1, and MDAPanc-28, in which TAK1 expression was silenced by small hairpin RNA. TAK1 kinase activity was targeted in AsPc-1, PANC-1, MDAPanc-28, and Colo357FG cells with exposure to increasing doses of a selective small-molecule inhibitor, LYTAK1, for 24 hours. To test the effect of LYTAK1 in combination with chemotherapeutic agents, AsPc-1, PANC-1, MDAPanc-28 cells, and control cells were treated with increasing doses of oxaliplatin, SN-38, or gemcitabine in combination with LYTAK1. In vivo activity of oral LYTAK1 was evaluated in an orthotopic nude mouse model (n=40, 5 per group) with luciferase-expressing AsPc-1 pancreatic cancer cells. Results AsPc-1 and MDAPanc-28 TAK1 knockdown cells had a statistically significantly lower NF-kappaB activity than did their respective control cell lines. TAK1 inhibitor LYTAK1 had potent in vitro cytotoxic activity in AsPc-1, PANC-1, MDAPanc-28, and Colo357FG cells, with IC50 between 5 and 40 nM. LYTAK1 also potentiated the cytotoxicity of chemotherapeutic agents oxaliplatin, SN-38, and gemcitabine in AsPc-1, PANC-1, and MDAPanc-28 cells compared with control cells. In nude mice, oral administration of LYTAK1 plus gemcitabine statistically significantly reduced tumor burden and prolonged survival duration. Conclusions The results of this study suggest that genetic silencing or inhibition of TAK1 kinase activity in vivo is a potential therapeutic approach to reversal of the intrinsic chemoresistance of pancreatic cancer., JOP. Journal of the Pancreas, Vol 14, N° 5S (2013): September (Suppl.) - p. 528-602

This dissertation addresses signaling mechanisms involved in thyroid carcinoma formation, with a view of identifying novel levels for therapeutic intervention. In this frame, appended manuscripts II, III and IV focus on the functional characterization of a novel BRAF mutation and on molecular targeting of RET or BRAF pathways in thyroid cancer. The main body of the dissertation describes a novel signaling mechanism that we have identified in thyroid carcinomas harboring BRAF or RET/PTC oncogenes and that involves CD44. CD44, a cell surface adhesion molecule overexpressed in a wide range of cancer types, undergoes sequential proteolytic cleavage at the extracellular and intramembrane domains. This results in the shedding of the ectodomain (ectoCD44) and in the intracellular release of an intracellular fragment (CD44-ICD). CD44-ICD is translocated to the nucleus and activates gene transcription. Our group has previously demonstrated that CD44 is overexpressed in papillary thyroid carcinoma (PTC) tissue samples and in cell lines harboring RET/PTC or BRAF oncogenes. Here, we show that RET/PTC signaling induces γ-secretase-dependent CD44 proteolysis through the ERK pathway. Chemical blockade of either RET/PTC or MEK abrogates CD44 cleavage. Adoptive overexpression of CD44-ICD stimulates TSH-independent proliferation of thyroid follicular PC cells. CD44-ICD binds to CREB and stimulates CREB-mediated transcription in PC cells by potentiating CREB S133 phosphorylation. Through this mechanism, CD44-ICD up-regulates cyclin D1 expression and proliferation of thyroid cells. Taken together, these findings suggest that ERK kinase pathway-mediated CD44 cleavage sustains proliferation of thyrocytes harboring RET/PTC, RAS or BRAF oncogenes. This novel pathway may provide new molecular targets for therapeutic intervention in thyroid carcinoma.

Here we describe a conditional doxycycline-dependent mouse model of RET/PTC3 (NCOA4-RET) oncogene-induced thyroid tumorigenesis. In these mice, after 10 days of doxycycline (dox) administration, RET/PTC3 expression induced mitogen activated protein kinase (MAPK) stimulation and a proliferative response which resulted in the formation of hyperplastic thyroid lesions. This was followed, after 2 months, by growth arrest accompanied by typical features of oncogene-induced senescence (OIS), including upregulation of p16INK4A and p21CIP, positivity at the Sudan black B, activation of the DNA damage response (DDR) markers γH2AX and pChk2 T68, and induction of p53 and p19ARF. After 5 months, about half of thyroid lesions escaped OIS and formed tumors that remained dependent on RET/ PTC3 expression. This progression was accompanied by activation of AKT-FOXO1/3a pathway and increased serum TSH levels.

Oncogenic conversion of the RET tyrosine kinase is a frequent feature of medullary thyroid carcinoma (MTC). ZD6474 (vandetanib) is an ATP-competitive inhibitor of RET, epidermal growth factor receptor (EGFR), and vascular endothelial growth factor receptors kinases. In this study, we have studied ZD6474 mechanism of action in TT and MZ-CRC-1 human MTC cell lines, carrying cysteine 634 to tryptophan (C634W) and methionine 918 to threonine (M918T) RET mutation respectively. ZD6474 blunted MTC cell proliferation and RET, Shc and p44/p42 mitogen-activated protein kinase (MAPK) phosphorylation. Single receptor knockdown by RNA interference showed that MTC cells depended on RET for proliferation. Adoptive expression of the ZD6474-resistant V804M RET mutant rescued proliferation of TT cells under ZD6474 treatment, showing that RET is a key ZD6474 target in these MTC cells. Upon RET inhibition, adoptive stimulation of EGFR partially rescued TT cell proliferation, MAPK signaling, and expression of cell-cycle-related genes. This suggests that simultaneous inhibition of RET and EGFR by ZD6474 may overcome the risk of MTC cells to escape from RET blockade through compensatory over-activation of EGFR.

Context: The V600E mutation accounts for the vast majority of thyroid carcinoma-associated BRAF mutations. Objective: The aim was to study the effects of the two BRAF V600E ATP-competitive kinase inhibitors, PLX4032 and PLX4720, in thyroid carcinoma cell lines. Experimental Design: We examined the activity of PLX4032 and PLX4720 in thyroid carcinoma cell lines harboring BRAF V600E (8505C, BCPAP, SW1736, BHT101), NRAS Q61R (HTH7), KRAS G12R (CAL62), HRAS G13R (C643), or RET/PTC1 (TPC-1) oncogenes. Normal thyrocytes (PC Cl 3) were used as control. Results: Both compounds inhibited the proliferation of BRAF mutant cell lines, but not normal thyrocytes, with a half maximal effective concentration (EC50) ranging from 78-113 nM for PLX4720 and from 29-97 nM for PLX4032. Doses equal to or higher than 500 nM were required to achieve a similar effect in BRAF wild-type cancer cells. Phosphorylation of ERK 1/2 and MAPK kinase (MEK) 1/2 decreased upon PLX4032 and PLX4720 treatment in BRAF mutant thyroid carcinoma cells but not in normal thyroid cells or in cell lines harboring mutations of RAS or RET/PTC1 rearrangements. PLX4032 and PLX4720 treatment induced a G1 block and altered expression of genes involved in the control of G1-S cell-cycle transition. 8505C cell tumor xenografts were smaller in nude mice treated with PLX4032 than in control mice. This inhibition was associated with reduction of phospho-ERK and phospho-MEK levels. Conclusions: This study provides additional evidence of the promising nature of mutant BRAF as a molecular target for thyroid carcinoma cells.