Galinos Fanourakis

Molecular sequelae of proteasome inhibition in human multiple myeloma cells

The proteasome inhibitor PS-341 inhibits IκB degradation, prevents NF-κB activation, and induces apoptosis in several types of cancer cells, including chemoresistant multiple myeloma (MM) cells. PS-341 has marked clinical activity even in the setting of relapsed refractory MM. However, PS-341-induced apoptotic cascade(s) are not yet fully defined. By using gene expression profiling, we characterized the molecular sequelae of PS-341 treatment in MM cells and further focused on molecular pathways responsible for the anticancer actions of this promising agent. The transcriptional profile of PS-341-treated cells involved down-regulation of growth/survival signaling pathways, and up-regulation of molecules implicated in proapoptotic cascades (which are both consistent with the proapoptotic effect of proteasome inhibition), as well as up-regulation of heat-shock proteins and ubiquitin/proteasome pathway members (which can correspond to stress responses against proteasome inhibition). Further studies on these pathways showed that PS-341 decreases the levels of several antiapoptotic proteins and triggers a dual apoptotic pathway of mitochondrial cytochrome c release and caspase-9 activation, as well as activation of Jun kinase and a Fas/caspase-8-dependent apoptotic pathway [which is inhibited by a dominant negative (decoy) Fas construct]. Stimulation with IGF-1, as well as overexpression of Bcl-2 or constitutively active Akt in MM cells also modestly attenuates PS-341-induced cell death, whereas inhibitors of the BH3 domain of Bcl-2 family members or the heat-shock protein 90 enhance tumor cell sensitivity to proteasome inhibition. These data provide both insight into the molecular mechanisms of antitumor activity of PS-341 and the rationale for future clinical trials of PS-341, in combination with conventional and novel therapies, to improve patient outcome in MM.

Novel Histone Deacetylase Inhibitors in the Treatment of Thyroid Cancer

Histone deacetylases (HDAC) and histone acetyltransferases exert opposing enzymatic activities that modulate the degree of acetylation of histones and other intracellular molecular targets, thereby regulating gene expression, cellular differentiation, and survival. HDAC inhibition results in accumulation of acetylated histones and induces differentiation and/or apoptosis in transformed cells. In this study, we characterized the effect of two HDAC inhibitors, suberoylanilide hydroxamic acid (SAHA) and m-carboxycinnamic acid bis-hydroxamide, on thyroid carcinoma cell lines, including lines originating from anaplastic and medullary carcinomas. In these models, both SAHA and m-carboxycinnamic acid bis-hydroxamide induced growth arrest and caspase-mediated apoptosis and increased p21 protein levels, retinoblastoma hypophosphorylation, BH3-interacting domain death agonist cleavage, Bax up-regulation, down-regulation of Bcl-2, A1, and Bcl-xL expression, and cleavage of poly(ADP-ribose) polymerase and caspase-8, -9, -3, -7, and -2. Transfection of Bcl-2 cDNA partially suppressed SAHA-induced cell death. SAHA down-regulated the expression of the apoptosis inhibitors FLIP and cIAP-2 and sensitized tumor cells to cytotoxic chemotherapy and death receptor activation. Our studies provide insight into the tumor type–specific mechanisms of antitumor effects of HDAC inhibitors and a framework for future clinical applications of HDAC inhibitors in patients with thyroid cancer, including histologic subtypes (e.g., anaplastic and medullary thyroid carcinomas) for which limited, if any, therapeutic options are available.

Targeting BRAFV600E in thyroid carcinoma: therapeutic implications

B-Raf is an important mediator of cell proliferation and survival signals transduced via the Ras-Raf-MEK-ERK cascade. BRAF mutations have been detected in several tumors, including papillary thyroid carcinoma, but the precise role of B-Raf as a therapeutic target for thyroid carcinoma is still under investigation. We analyzed a panel of 93 specimens and 14 thyroid carcinoma cell lines for the presence of BRAF mutations and activation of the mitogen-activated protein/ERK kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway. We also compared the effect of a B-Raf small inhibitory RNA construct and the B-Raf kinase inhibitor AAL881 on both B-Raf wild-type and mutant thyroid carcinoma cell lines. We found a high prevalence of the T1799A (V600E) mutation in papillary and anaplastic carcinoma specimens and cell lines. There was no difference in patient age, B-Raf expression, Ki67 immunostaining, or clinical stage at presentation between wild-type and BRAFV600E specimens. Immunodetection of phosphorylated and total forms of MEK and ERK revealed no difference in their phosphorylation between wild-type and BRAFV600E patient specimens or cell lines. Furthermore, a small inhibitory RNA construct targeting the expression of both wild-type B-Raf and B-RafV600E induced a comparable reduction of viability in both wild-type and BRAFV600E mutant cancer cells. Interestingly, AAL881 inhibited MEK and ERK phosphorylation and induced apoptosis preferentially in BRAFV600E-harboring cells than wild-type ones, possibly because of better inhibitory activity against B-RafV600E. We conclude that B-Raf is important for the pathophysiology of thyroid carcinomas irrespective of mutational status. Small molecule inhibitors that selectively target B-RafV600E may provide clinical benefit for patients with thyroid cancer. [Mol Cancer Ther 2007;6(3):1070–8]

Fas Signaling in Thyroid Carcinomas Is Diverted from Apoptosis to Proliferation

Purpose: The death receptor Fas is present in thyroid carcinomas, yet fails to trigger apoptosis. Interestingly, Fas has been reported to be actually overexpressed in papillary thyroid carcinomas, suggesting that it may confer a survival advantage. Experimental Design: We investigated the expression and activation status of Fas pathway mediators in thyroid carcinoma cell lines and tumor specimens. Results: All cell lines tested express Fas-associated death domain, procaspase-8, procaspase-9, and procaspase-3; resistance to Fas-mediated apoptosis could not be attributed to lack of any of these apoptosis mediators. Moreover, Fas death domain mutations were not found in our study. The proteasome inhibitors MG132 and PS-341 (bortezomib, Velcade), which lead to accumulation of the nuclear factor κB (NF-κB) inhibitor IκB, did not sensitize SW579 cells to Fas-mediated apoptosis, suggesting that resistance to Fas-mediated apoptosis is not due to proteasome or NF-κB activity. Cross-linking of Fas in vitro induced recruitment of Fas-associated death domain–like interleukin-1β–converting enzyme inhibitory protein (FLIP) instead of procaspase-8. Inhibition of FLIP expression with a FLIP antisense oligonucleotide resulted in significant sensitization to Fas-mediated apoptosis. Fas cross-linking promoted BrdUrd incorporation; activated the mitogen-activated protein kinase/extracellular signal–regulated kinase kinase/extracellular signal–regulated kinase, NF-κB, and activator protein-1 pathways in thyroid carcinoma cells in vitro; and protected cells from tumor necrosis factor–related apoptosis-inducing ligand–induced apoptosis. We also found that good prognosis papillary thyroid carcinoma specimens exhibited higher immunoreactivity for cleaved (activated) caspase-8 than poor prognosis tumors. Conclusions: In thyroid carcinomas, the proteolytic cleavage and activation of caspase-8 depends on the balance between expression levels for procaspase-8 and FLIP and correlates with favorable clinical prognosis. Fas may actually stimulate proliferation and confer a survival advantage to thyroid cancer cells.

Mutational analysis of the BRAF, RAS and EGFR genes in human adrenocortical carcinomas

The serine/threonine kinase B-Raf plays a key role in the Ras/Raf/MEK/ERK pathway that relays extracellular signals for cell proliferation and survival. Several types of human malignancies harbor activating BRAF mutations, most frequently a V600E substitution. The epidermal growth factor receptor (EGFR), a transmembrane tyrosine kinase (TK) receptor that mediates proliferation and survival signaling, is expressed in a wide variety of normal and neoplastic tissues. EGFR inhibitors have produced objective responses in patients with non-small cell lung carcinomas harboring activating EGFR TK domain somatic mutations. We evaluated the presence of mutations in BRAF (exons 11 and 15), KRAS (exons 1 and 2), NRAS (exons 1 and 2), and EGFR (exons 18-21) in adrenal carcinomas (35 tumor specimens and two cell lines) by DNA sequencing. BRAF mutations were found in two carcinomas (5.7%). Four carcinomas (11.4%) carried EGFR TK domain mutations. One specimen carried a KRAS mutation, and another carried two NRAS mutations. No mutations were found in the two adrenocortical cell lines. BRAF- and EGFR-mutant tumor specimens exhibited stronger immunostaining for the phosphorylated forms of the MEK and ERK kinases than their wild-type counterparts. EGFR-mutant carcinomas exhibited increased phosphorylation of EGFR (Tyr 992) compared with wild-type carcinomas. We conclude that BRAF, RAS, and EGFR mutations occur in a subset of human adrenocortical carcinomas. Inhibitors of the Ras/Raf/MEK/ERK and EGFR pathways represent candidate targeted therapies for future clinical trials in carefully selected patients with adrenocortical carcinomas harboring respective activating mutations.

Atherosclerosis of the Carotid Artery: Absence of Evidence for CMV Involvement in Atheroma Formation

Several studies suggest that certain viral and bacterial pathogens may contribute to the process of atherogenesis. However, this relation between infectious agents and atherosclerosis has not yet been established with certainty. The aim of this study was to investigate the presence of CMV in carotid endarterectomies from 40 patients suffering from atherosclerosis using immunohistochemistry and the polymerase chain reaction (PCR). None of the specimens examined gave a positive result, indicating absence of CMV particles or CMV DNA sequences in the walls of carotid arteries. This finding suggests it is possible that CMV infection may not play a major role in the formation of atheroma. Therefore, further investigation is required in order to clarify the etiology of atherosclerosis.

Expression of MMPs and TIMP-1 in smoker and nonsmoker chronic periodontitis patients before and after periodontal treatment.

BACKGROUND AND OBJECTIVE Nonsurgical periodontal treatment controls periodontal inflammation. Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are implicated both in the destruction and in the healing of periodontal tissues. The aim of the present study was to compare the mRNA expression of MMP-1, -3, -8, -9 and -13 and TIMP-1 in chronic periodontitis before and after initial periodontal treatment. MATERIAL AND METHODS Ninety gingival samples were harvested from 30 patients with chronic periodontitis (15 nonsmokers and 15 smokers) before and after nonsurgical treatment and from 30 periodontally healthy control subjects (15 nonsmokers and 15 smokers). Clinical parameters were assessed before and after treatment. Total RNA was isolated, and mRNA expression of MMPs and TIMP-1 was assessed by RT-PCR. RESULTS Periodontal treatment significantly increased TIMP-1 expression and decreased the ratios of MMPs/TIMP-1. Post-treatment, nonsmokers with periodontitis had significantly higher MMP-8 and TIMP-1 expression than healthy nonsmokers, and smokers with periodontitis had significantly higher MMP-13 and TIMP-1 expressions than healthy smokers. Post-treatment, smokers had significantly higher TIMP-1 expression and lower MMP-8/TIMP-1 ratio than nonsmokers. Post-treatment, there was no correlation among MMPs, and the expression of MMPs and TIMP-1 was not correlated with clinical measurements. CONCLUSION Periodontal treatment increased TIMP-1 expression and decreased the ratios of MMPs/TIMP-1 in chronic periodontitis. The post-treatment increase in TIMP-1 expression was higher for smokers. The TIMP-1 expression was higher post-treatment than in health. Post-treatment, MMP-8 expression was higher in nonsmokers with periodontitis than in healthy nonsmokers, whereas MMP-13 expression was higher in smokers with periodontitis than in healthy smokers.

The effect of smoking on the mRNA expression of MMPs and TIMP-1 in untreated chronic periodontitis patients: a cross-sectional study.

BACKGROUND AND OBJECTIVE Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are important for extracellular matrix. Expression of MMPs has been evaluated in gingiva without studying smoking. The aim of this study was to explore the effect of smoking on mRNA expression of MMP-1, -3, -8, -9 and -13 and TIMP-1 in untreated chronic periodontitis and in periodontal health. MATERIAL AND METHODS Gingival samples were harvested from 30 subjects with untreated chronic periodontitis (15 nonsmokers and 15 smokers) and 30 periodontally healthy subjects (15 nonsmokers and 15 smokers). Full-mouth plaque score, gingival index, bleeding on probing, probing depth and clinical attachment level were recorded. Total RNA was isolated, and the mRNA expression of MMPs and TIMP-1 was assessed by RT-PCR. RESULTS Periodontitis groups were comparable in clinical measurements. Nonsmoker subjects with periodontitis had statistically significantly higher MMP-1, lower MMP-9 and TIMP-1 expression and higher MMP-1/TIMP-1 ratio than smokers; and higher MMP-8 expression and MMP-8/TIMP-1 and MMP-1/TIMP-1 ratios than healthy nonsmokers. Healthy nonsmokers had statistically significantly higher MMP-13 expression than healthy smokers. Smoker periodontitis and healthy subjects had similar expression levels of MMPs and TIMP-1 and MMPs/TIMP-1 ratios. There was correlation among the MMPs only for smoker periodontitis subjects. Expression of MMP-13 was correlated with mean clinical attachment level. CONCLUSION Within its limits, this study demonstrated that smoking affected mRNA expression of MMPs and TIMP-1, MMPs/TIMP-1 ratios and relationships among MMPs in untreated chronic periodontitis and expression of MMPs in health. In the absence of smoking, chronic periodontitis affected expression of MMPs and MMPs/TIMP-1 ratios.

Expression of receptor activator of NF-κB ligand and osteoprotegerin in peripheral giant cell granulomas of the jaws.

BACKGROUND Peripheral giant cell granuloma is a tumor of the jaw characterized by the presence of multinucleated giant cells and mononuclear cells within a fibrous stroma. These lesions are considered to be of a reactive nature rather than neoplastic. Although peripheral giant cell granulomas is a well-described clinical entity, little is known on its pathogenesis. The aim of this study was to investigate the receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) expression and immunolocalization in giant cell granulomas. METHODS RANKL and OPG protein expression was evaluated in 22 peripheral giant cell granulomas samples, by means of immunohistochemistry. Staining was evaluated semi-quantitatively, according to the extent and intensity of the stain. RESULTS RANKL was expressed in all cases with a cytoplasmic staining pattern, whereas OPG expression was detected in 21 of the 22 cases examined. Active multinucleated giant cells exhibited intense immunoreactivity for both proteins. CONCLUSION RANKL and OPG are expressed in peripheral giant cell granulomas of the jaw in a manner supporting the osteoclastic nature of giant cells whereas the possible osteoclastic lineage of stromal monocytes remains ambiguous.

Fibrillin expression and localization in various types of carcinomas of the thyroid gland

Fibrillin is an extracellular matrix (ECM) glycoprotein, a main component of microfibrills, suggested to support cell attachment and to impact cell differentiation and migration. The aim of this study was to investigate fibrillin-1 expression in thyroid carcinomas at mRNA and protein level, since ECM proteins are suggested to be of great importance for the metastatic potential of carcinomas. RNA was extracted from 13 thyroid carcinoma cell lines and RT-PCR analysis with gene-specific primers revealed fibrillin-1 mRNA expression in all cell lines, with highest expression in the follicular carcinoma cell line WRO and lowest expression in the two anaplastic cell lines (APO, FRO). Furthermore, we investigated fibrillin-1 expression by immumohistochemistry in a commercially available tissue microarray including 50 thyroid carcinomas as well as in archival tissue from 33 thyroid carcinomas. Fibrillin-1 demonstrated a cytoplasmic location in the neoplastic cells of almost all carcinomas apart from the follicular ones. The most intense staining was observed in papillary carcinomas with some evidence of a slight increased intensity in advanced stages. Our data indicate that fibrillin-1 is strongly expressed by the neoplastic cells of thyroid carcinomas in different degree in the various histologic types and might be implicated in cell–stroma interaction in terms of signaling, attachment and migration.