New molecular insights about hepatocellular carcinoma behaviour and the control of cell progression

2007/2008Studio dei fattori di elongazione eucariotici EEF1A e ruolo nello sviluppo dell'epatocarcinoma. Studio del bortezomib, inibitore del proteasoma, come potenziale farmaco da impiegare per il trattamento dell'epatocarcinoma.XXI Ciclo197

The Potential of MLN3651 in Combination with Selumetinib as a Treatment for Merlin-Deficient Meningioma

Meningioma is the most common primary intracranial tumour, and surgical resection is the main therapeutic option. Merlin is a tumour suppressor protein that is frequently mutated in meningioma. The activity of the E3 ubiquitin ligase complex, CRL4-DCAF1, and the Raf/MEK/ERK scaffold protein Kinase suppressor of Ras 1 (KSR1) are upregulated in Merlin-deficient tumours, which drives tumour growth. Identifying small molecules that inhibit these key pathways may provide an effective treatment option for patients with meningioma. We used meningioma tissue and primary cells derived from meningioma tumours to investigate the expression of DDB1 and Cullin 4-associated factor 1 (DCAF1) and KSR1, and confirmed these proteins were overexpressed. We then used primary cells to assess the therapeutic potential of MLN3651, a neddylation inhibitor which impacts the activity of the CRL family of E3 ubiquitin ligases and the MAPK/ERK kinase (MEK1/2) inhibitor selumetinib. MLN3651 treatment reduced proliferation and activated apoptosis, whilst increasing Raf/MEK/ERK pathway activation. The combination of MLN3651 and the MEK1/2 inhibitor selumetinib prevented the increase in Raf/MEK/ERK activity, and had an additive effect compared with either treatment alone. Therefore, the combined targeting of CRL4-DCAF1 and Raf/MEK/ERK activity represents an attractive novel strategy in the treatment of Merlin-deficient meningioma

β2-adrenoreceptor Signaling Increases Therapy Resistance in Prostate Cancer by Upregulating MCL1

There is accumulating evidence that continuous activation of the sympathetic nervous system due to psychosocial stress increases resistance to therapy and accelerates tumor growth via β2-adrenoreceptor signaling (ADRB2). However, the effector mechanisms appear to be specific to tumor type. Here we show that activation of ADRB2 by epinephrine, increased in response to immobilization stress, delays the loss of MCL1 apoptosis regulator (MCL1) protein expression induced by cytotoxic drugs in prostate cancer cells; and thus, increases resistance of prostate cancer xenografts to cytotoxic therapies. The effect of epinephrine on MCL1 protein depended on protein kinase A (PKA) activity, but was independent from androgen receptor expression. Furthermore, elevated blood epinephrine levels correlated positively with an increased MCL1 protein expression in human prostate biopsies. In summary, we demonstrate that stress triggers an androgen-independent antiapoptotic signaling via the ADRB2/PKA/MCL1 pathway in prostate cancer cells. IMPLICATIONS: Presented results justify clinical studies of ADRB2 blockers as therapeutics and of MCL1 protein expression as potential biomarker predicting efficacy of apoptosis-targeting drugs in prostate cancer

CBMT-06. HIGH LEVELS OF GATA-4 IN MALIGNANT MENINGIOMA LEAD TO LOWER miR-497~195 CLUSTER EXPRESSION IN TISSUE AND BLOOD

Abstract Meningioma is the most common primary intracranial brain tumour, classified as benign (WHO I, 70%), atypical (WHO II, 30%) and anaplastic/malignant (WHO III, 1–3%). The 3-year recurrence rate for WHO I tumours is 40% and it is much higher in WHO II-III1. To date, meningioma classification is based only on histopathological characterization, and no circulating biomarkers have been identified to predict tumour progression2. Indeed, microRNAs are promising circulating biomarkers because they can be released from tumour cells into the blood stream via exosomes, showing potential to be used as liquid biopsies3. Previously, Cyclin D1 overexpression was suggested as a possible meningioma biomarker as it correlates with proliferative activity and tumour grade, while high levels of Cyclin E1 were shown to correlate with higher recurrence rates4,5. In this study, we showed, for the first time, that the transcription factor GATA-4 is overexpressed in malignant meningioma cells and tissues, controlling the miR-15 family (miR-15a/b-16-497-195) expression. Moreover, we proved that the miR-497~195 cluster not only regulates Cyclin D1-D2-D3 and E1 levels, but it also modulates GATA-4 expression in meningioma. Moreover, we showed that the levels of miR-497~195 cluster are significantly lower in exosomes derived from higher grade meningioma patients’ blood serum, supporting their potential as diagnostic biomarkers.</jats:p

Abstract 183: Loss of Bad phosphorylation and Mcl-1 expression is necessary for rapid apoptosisinduced by combination of TGFα-PE and ZSTK474 in prostate cancer cells

Abstract Advanced androgen-independent prostate cancer is notoriously resistant to conventional systemic therapies, and at the moment there is no effective protocol for hormone-refractory advanced prostate cancer affected patients. One of possible mechanisms of such resistance is activation of the anti-apoptotic signaling PI3K/Akt pathway that is constitutively active in the 60% of advanced prostate cancers. However, multiple inhibitors of PI3K are just well studied and some have just gone on to clinical trials, but unfortunately they showed limited efficacy against prostate cancer alone. Our recent experiments has shown remarkable synergy in inducing rapid and massive apoptosis when PI3K inhibitor ZSTK474 is used in combination with Pseudomonas aeruginosa exotoxin A fragment fused with Transforming Growth Factor Alpha (TGFα-PE38). Time lapse video microscopy of prostate cancer C4-2 cell line has shown the substantial cell death within 4-6 hours with combined administration, compared to limited cell death in single agents-treated cells. Analysis of PARP and cleaved-caspase 3 fragment by Western blotting confirmed that cell death occurs via apoptosis. Quantitation of caspase 3 and 7 activity with luminescent AFC-DEVD substrate confirmed synergy in apoptosis activation by combination of TGFα-PE38 and ZSTK474, while single agents even in higher concentration did not induce substantial apoptosis within 6 hours time period. Analysis of the mechanisms underlying this synergy has shown that PI3K inhibitor ZSTK474 triggers Bad dephosphorylation, while TGFα-PE38 reduces expression levels of Mcl-1. A slight increase of Bim protein was detected and related with ZSTK474 administration. No major variations were detected in the expression levels of pro- and anti-apoptotic proteins Bax, Bcl-2 and Bcl-XL. To address the role of Bad phosphorylation and Mcl-1 expression in apoptosis induction by PI3K inhibitor ZSTK474 and TGFα-PE38, we examined apoptosis in C4-2 cells that express phosphorylation-deficient mutant Bad (BAD2SA). Results indicated that the expression of BAD2SA sensitized C4-2 cells to apoptosis induced by TGFα-PE38. Experiments that address the role of Mcl-1 loss in apoptosis induced by TGFα-PE38 are ongoing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 183. doi:10.1158/1538-7445.AM2011-183</jats:p

Abstract 2748: Prostate-specific inhibition of PI3 Kinase and protein synthesis as a therapy for advanced hormone-refractory prostate cancer

Abstract Prostate cancer remains the second leading cause of cancer-related death in men in the United States, as at present there are no effective treatments for advanced stage-affected patients. Recurrence usually involves hormonal androgen suppression, which typically translates into androgen-ablation insensitivity with accompanied limited or transient response to systemic chemotherapy. Our preliminary experiments have shown that it is possible to induce massive and rapid apoptosis in the hormone-refractory prostate cancer cells in vitro and in vivo, by using the PI3K inhibitor ZSTK474 in combination with a prostate cancer-selective cytotoxin J591PE38, a fusion protein between two fragments: one from the J591 antibody (that recognizes the Prostate-Specific Membrane Antigen, expressed in nearly all prostate cancers with the highest rate in poorly differentiated, metastatic and hormone-refractory cases), and the other from the Pseudomonas aeruginosa exotoxin A (PE38, that ADP-ribosylates and inhibits the Eukaryotic Elongation Factor 2). The PI3K inhibitor ZSTK474 in combination with the cytotoxin J591PE38 induces massive cell death (around 100%) in prostate cancer C4-2tetLuc and C4-2 cells monitored by Time-Lapse Video Microscopy during 24 hours, when compared to controls. Combined agents at 6 hours synergistically increases Caspase 3 activity, as confirmed by DEVD-Afc enzymatic assay (about 25 and 30 folds in C4-2tetLuc and C4-2 cells respectively, compared to control) and Western blotting (3.28 and 2.45 folds in C4-2tetLuc and C4-2 cells respectively, compared to control). Apoptosis activation was confirmed by detecting the cleaved fragments of Caspase 7 and PARP (compared to control respectively: 4.12 and 3.58 folds for C4-2tetLuc; 5.38 and 13.38 folds for C4-2 cells). No any major effects were found in the PSMA-negative prostate cancer PC-3 and breast cancer BT-549 cells treated with both agents when compared to controls, confirming the specificity of the cytotoxin J591PE38. PI3 Kinase inhibition by ZSTK474 was confirmed in all cell lines used by monitoring the phosphorylation levels of Akt (Thr308) by Western blotting. Prostate cancer C4-2tetLuc xenografts in nude mice showed a potent reduction of luminescence (around 95% at day 7) in tumors treated with a single local injection of both agents, when compared to controls. Apoptosis activation and PI3 Kinase inhibition at 6 hours were confirmed by monitoring the protein levels of cleaved-PARP and the phosphorylation levels of Akt by Western blotting. In summary, in this study we demonstrated the efficacy of a potential combinatorial approach using a specific PI3 Kinase and de novo protein synthesis inhibitor to treat hormone-refractory prostate cancer with constitutive active PI3K/Akt pathway, by generating massive and rapid apoptosis in vitro and in vivo (This work is supported by NIH/NCI grant 3R01 CA 118329 02S2). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2748. doi:1538-7445.AM2012-2748</jats:p

Bortezomib effect of E2F and cyclin family members in human hepatocellular carcinoma cell lines

AIM To evaluate the effects of the proteasome inhibitor Bortezomib (BZB) on E2Fs and related genes in Hepatocellular carcinoma (HCC) cells. METHODS The mRNA levels of the E2F family members (pro-proliferative: E2F1-3 and anti-proliferative: E2F4-8) and of their related genes cyclins and cyclin-dependent kinases (cdks) were evaluated in two HCC cell lines following a single BZB administration. mRNA levels of the epithelial-mesenchymal transition (EMT) genes were also measured in both cell lines after BZB treatment. The BZB concentration (40 nM) used was chosen to stay well below the maximal amount/cm2 recommended for in vivo application, and two days incubation was chosen as this time point has been found optimal to detect BZB effects in our previous studies. The HCC cell lines, HepG2 and JHH6, were chosen as they display different phenotypes, hepatocyte-like for HepG2 and undifferentiated for JHH6, thus representing an in vitro model of low and high aggressive forms of HCC, respectively. The mRNA levels of the target genes were measured by two-color microarray-based gene expression analysis, performed according to Agilent Technologies protocol and using an Agilent Scan B. For the E2F family members, mRNA levels were quantified by RT-PCR. Using small interfering RNA\u2019s, the effects of E2F8 depletion on cell number was also evaluated. RESULTS After BZB treatment, microarray analysis of the undifferentiated JHH6 revealed a significant decrease in the expression of the pro-proliferative E2F member E2F2. Quantitative RT-PCR data were in keeping with the microarray analysis, and showed a significant increase and decrease in E2F8 and E2F2 mRNA levels, respectively. In contrast, BZB treatment of the hepatocyte-like HCC cell line HepG2 had a significant impact on mRNA levels of 5 of the 8 E2F members. In particular, mRNA levels of the pro-proliferative E2F members E2F1, E2F2, and of the anti-proliferative member E2F8, decreased over 80%. Notably, a reduction in E2F8 expression in HepG2 and JHH6 cells following siRNA treatment had no impact on cell proliferation. As observed with JHH6, BZB treatment of HepG2 cells induced a significant increase in mRNA levels of an anti-proliferative E2F member, E2F6 in this case. As was observed with E2F\u2019s, more dramatic changes in mRNA levels of the E2F related genes cyclins and Cdks and the epithelial-mesenchymal transition (EMT) genes were observed after BTZ treatment of HepG2 compared to JHH6 . CONCLUSION The differential expression of E2Fs and related genes induced by BZB in diverse HCC cell phenotypes contribute to bortezomib\u2019s mechanism of action in hepatocellular carcinoma

A pilot study to establish non-invasive biomarkers for higher-grade meningioma

Abstract Introduction Meningioma brain tumours are the most common primary tumour in adults. Despite surgery and/or radiation therapy, meningioma may recur. The 5-year recurrence rate in benign meningioma is estimated in about 10% while much greater in atypical and malignant tumours. MicroRNAs (miRNAs) represent a large class of small RNAs driving regulation of gene expression and playing a role in tumour progression and therefore proposed as diagnostic tools. Moreover, miRNAs can be released from tumour cells into the blood stream via exosomes, showing potential to be used as liquid biopsies. Methods Identification of novel circulating biomarkers was conducted by performing an unbiased Cancer MicroRNA qPCR Array, followed by bioinformatics analysis. In parallel, we conducted a biased in silico analysis of the miRNAs targeting Cyclin D1 and E1, recently proposed as immunohistochemical meningioma biomarkers. Validation studies performed using TaqMan® reagents. Results Stringent unbiased (p&lt;0.01) miRNA profiling followed by validation in ex vivo samples revealed that the miR-9-1 is upregulated in higher-grade meningioma tissues and serum exosomes, controlled by the EGFR/AP-1 axis and correlated with lower levels of E-Cadherin, a proposed biomarker for malignant meningioma. On the contrary, biased analysis, followed by validation in vitro and ex vivo, showed that the miR-497~195 cluster is downregulated in higher-grade meningioma tissues and serum exosomes, correlating with the overexpression of GATA-4, a novel meningioma tissue biomarker. Conclusion Our data demonstrated that both miR-497~195 and miR-9-1 show potential to become promising non-invasive biomarkers for higher-grade meningioma, reflecting their expression status in tissues. (DB and CN contributed equally). </jats:sec