Alfonso Catalano

Vascular endothelial growth factor is an autocrine growth factor in human malignant mesothelioma

Vascular endothelial growth factor (VEGF), a potent mitogen for vascular endothelium, is expressed in malignant pleural mesothelioma (MM). The present report examines the effect of VEGF on MM growth. Four MM cell lines produced significantly higher VEGF levels than normal mesothelial cells (1946±14 pg/ml vs. 180±17 pg/ml; p<0.001). In addition, MM cells expressed the tyrosine kinase‐related VEGF receptors Flt‐1 and KDR. Recombinant human VEGF phosphorylated both Flt‐1 and KDR and increased proliferation of all four MM cell lines in a dose‐dependent fashion. Neutralizing antibodies against either VEGF, Flt‐1 or KDR significantly reduced MM cellular proliferation. In addition, expression of VEGF, Flt‐1, and KDR was observed in MM biopsies. Moreover, higher VEGF levels were found in the pleural effusions of MM patients than in the effusions of patients with non‐malignant pleural disease (1885.7±894.9 pg/ml vs. 266.9±180.5 pg/ml; p<0.001). Linear regression analysis showed a significant inverse correlation between serum VEGF levels and MM patient survival (r=0.72; p<0.01). No correlation was found between tumour vessel density and either serum (r=0.26; p=0.42) or pleural effusion (r=0.35; p=0.26) VEGF levels. These results indicate that VEGF, via activation of its tyrosine kinase receptors, may be a key regulator of MM growth. In addition, VEGF production could have an impact on patient survival, not only by promoting tumour angiogenesis but also by directly stimulating tumour growth. Copyright

FLIP overexpression inhibits death receptor-induced apoptosis in malignant mesothelial cells

Tumors have developed several forms of resistance to receptor-induced cell death. Here, we show that malignant mesothelial (MM) cell lines as well as primary MM cells and normal mesothelial (NM) cells express Fas and TNF-related apoptosis-inducing ligand (TRAIL) receptors DR4 and DR5. We found that, although Fas expression levels are comparable, only MM cells are resistant to cell death. Furthermore, MM cells show resistance to TRAIL-induced apoptosis. Caspase-8 (FLICE) is not activated by death receptors triggering in malignant cells whereas it is well activated by nonreceptor stimuli, such as UV radiation. We found that FLIP (FLICE-Inhibitory Protein) is constitutively expressed in all MM cell lines and is more expressed in primary MM cells than in NM cells. Knockdown of FLIP expression in MM cell lines, by a FLIPsiRNA, re-established the normal response to apoptosis induced by Fas or DR4/DR5, which was blocked by pretreatment with the caspase-8 inhibitor z-IETD-fmk. These results indicate that MM cells develop an intrinsic resistance to apoptosis induced by death receptors upregulating the expression of the antiapoptotic protein c-FLIP.

5-Lipoxygenase regulates senescence-like growth arrest by promoting ROS-dependent p53 activation

5‐Lipoxygenase (5LO) is involved in the production of leukotrienes and reactive oxygen species (ROS) from arachidonic acid. Its strong activation has been associated with several diseases like cancer and neurodegeneration. Here we show that 5LO activity increases during senescence‐like growth arrest induced by oncogenic ras or culture history in both human and mouse embryo fibroblasts. Overexpression of 5LO promotes senescence‐like growth arrest via a p53/p21‐dependent pathway, and this occurs independently of telomerase activity. 5LO stabilizes p53 through phosphorylation at Ser15 and increases expression of the p53‐transcriptional target p21. This is achieved by regulating ROS production. Indeed, ROS are increased in 5LO‐arrested cells. Antioxidants and a low oxygen environment prevent 5LO‐induced growth arrest. Finally, 5LO inhibition reduces the growth arrest induced by oncogenic ras or culture history and these effects are neutralized by the addition of exogenous ROS. These data link the 5LO pathway to oxidative crises of primary fibroblast and suggest that the ability of 5LO to induce senescence‐like growth arrest may be important in the pathogenesis of 5LO‐associated disorders.

Cross-talk between vascular endothelial growth factor and semaphorin-3A pathway in the regulation of normal and malignant mesothelial cell proliferation

Vascular endothelial growth factor (VEGF) and semaphorin‐3A (Sema‐3A) play important roles in the transduction of promitotic and antimitotic signals, respectively. Here, we report that these conflicting signals are integrated via negative feedback between VEGF and Sema‐3A pathways in several primary normal, but not malignant, mesothelial cells. Unlike malignant mesothelial (MM) cells, in which VEGF induces cell proliferation, normal mesothelial (NM) cell growth was repressed by VEGF. Although both cell‐types expressed an overlapping set of VEGF tyrosine‐kinase receptors, only in NM cells VEGF exposure entails a p38 mitogen‐activated protein kinase (MAPK)‐dependent increased of Sema‐3A production. Inhibition of p38 MAPK (by SB202190 and SB203580) or a dominant‐negative mutant of Sema‐3A receptor plexin‐A1 reversed the inhibitory effects of VEGF in NM cells, increasing cyclin D1 synthesis and cell growth. Conversely, sustained activation of p38 MAPK by the p38 MAPK‐activating kinases MKK3 and MKK6 or transfection with Sema‐3A inhibited VEGF‐induced cyclin D1 up‐regulation and MM cell proliferation. Therefore, these results delineate a new role of Sema‐3A in VEGF function mediated by p38 MAPK and suggest that the abrogation of regulated Sema‐3A expression is responsible for VEGF‐driven growth of tumor cells.

Methionine Aminopeptidase-2 Regulates Human Mesothelioma Cell Survival: Role of Bcl-2 Expression and Telomerase Activity

Methionine aminopeptidase-2 (MetAP2) is the molecular target of the angiogenesis inhibitors, fumagillin and ovalacin. Fumagillin can also inhibit cancer cell proliferation, implying that MetAP2 may play a quite complex role in tumor progression. Here, we examined the expression and function of MetAP2 in an in vitro model of human mesothelioma. We found that mesothelioma cells expressed higher MetAP2 mRNA levels than primary normal mesothelial cells. Consistently, fumagillin induced apoptosis, owing to early mitochondrial damage, in malignant, but not in normal mesothelial cells. Transfection of mesothelioma cells with a MetAP2 anti-sense oligonucleotide determined a time-dependent inhibition of cell survival and induced nucleosome formation. Interestingly, mRNA and protein levels of the anti-apoptotic gene bcl-2 as well as telomerase activity were selectively reduced after MetAP2 inhibition in mesothelioma cells, whereas bcl-2 overexpression counteracted the effect of MetAP2 inhibition on telomerase activity and apoptosis. MetAP2 inhibition also increased caspase activity and the caspase inhibitor, zVAD-fmk, prevented fumagillin-induced apoptosis, but it did not alter telomerase activity. These results indicate that MetAP2 is a main regulator of proliferative and apoptotic pathways in mesothelioma cells and suggest that MetAP2 inhibition may represent a potential target for therapeutic intervention in human mesothelioma.

Enhanced expression of vascular endothelial growth factor (VEGF) plays a critical role in the tumor progression potential induced by simian virus 40 large T antigen.

Vascular endothelial growth factor (VEGF), an important angiogenic factor, regulates cell proliferation, differentiation, and apoptosis through activation of its tyrosine-kinase receptors, such as Flt-1 and Flk-1/Kdr. Human malignant mesothelioma cells (HMC), which have wild-type p53, express VEGF and exhibit cell growth increased by VEGF. Here, we demonstrate that early transforming proteins of simian virus (SV) 40, large tumor antigen (Tag) and small tumor antigen (tag), which have been associated with mesotheliomas, enhanced HMC proliferation by inducing VEGF expression. SV40-Tag expression potently increased VEGF protein and mRNA levels in several HMC lines. This effect was suppressed by the protein synthesis inhibitor, cycloheximide. Inactivation of the VEGF signal transduction pathway by expression of soluble form of Flt-1 inhibited Flk-1/Kdr activation and HMC proliferation induced by SV40 early genes. Experiments with SV40 mutants revealed that SV40-Tag, but not -tag, is involved in the VEGF promoter activation. However, concomitant expression of SV40-tag enhanced Tag function. In addition, SV40-Tag expression sustained VEGF induction in colon carcinoma cell line (CCL)-233, which have wild-type p53, but not in CCL-238, which lack functional p53. These data indicate that VEGF regulation by SV40 transforming proteins can represent a key event in SV40 signaling relevant for tumor progression.

Preclinical evaluation of the nonsteroidal anti-inflammatory agent celecoxib on malignant mesothelioma chemoprevention

Malignant mesothelioma (MM) remains the most lethal pleural, peritoneal and pericardial cancer. Here, we characterize the effects of nonsteroidal anti‐inflammatory agents (NSAIDs) on in vitro and in vivo experimental MM models. Unlike primary normal mesothelial cells, the selective cyclooxygenase (COX)‐2 inhibitor celecoxib reduced the in vitro proliferation of several MM cells derived from previously untreated MM patients. Moreover, celecoxib significantly inhibited MM cell colony formation in soft agarose (63–78% at 5 × 10−5 M; p ≤ 0.05) and it elicited remarkable antitumor activity, leading to long‐term survival in >37% of nude mice bearing intraperitoneal MM. Celecoxib was more efficient in inhibiting MM cell growth than acetylsalicylic acid (10−6 M‐10−2 M), indometacin (10−6 M‐10−2 M) and the COX‐2 inhibitor NS‐398 (10−6 M‐10−4 M). Efficacy of these different compounds was not related to the amount of COX‐2 protein levels present on MM cells. Celecoxib, in a dose‐ and time‐dependent manner, induced MM cell apoptosis, which involved decreased Akt phosphorylation, loss of Bcl‐2 and Survivin protein expression and caspase‐3 activation. Furthermore, vascular endothelial growth factor (VEGF), an MM autocrine growth factor and Akt inducer, rescued celecoxib‐induced apoptosis and Akt dephosphorylation. When the VEGF receptor (KDR/Flk‐1) inhibitor, SU‐1498, was used in combination with celecoxib, IC50 of celecoxib in vitro was reduced up to 65%. These data demonstrate that celecoxib may have antitumor properties in MM and provide a rationale for the therapeutic use of celecoxib in combination with a selective VEGF inhibitor.

The Plexin-A1 Receptor Activates Vascular Endothelial Growth Factor-Receptor 2 and Nuclear Factor-κB to Mediate Survival and Anchorage-Independent Growth of Malignant Mesothelioma Cells

The semaphorins and their receptors, the neuropilins and the plexins, are constituents of a complex regulatory system that controls axonal guidance. Moreover, many types of tumor cells express various members of semaphorins and receptors, but the biological activities within tumor mass and the signal transduction mechanism(s) they use are largely unknown. Here, we show that in asbestos-related malignant pleural mesothelioma (MPM), Semaphorin-6D (Sema6D) and its receptor plexin-A1 are frequently expressed and trigger a prosurvival program that promotes anchorage-independent growth of MPM cells. Interestingly, the same response is also controlled by the tyrosine kinase receptors of vascular endothelial growth factor (VEGF) through a nuclear factor-kappaB (NF-kappaB)-dependent pathway. We found that in MPM cells, plexin-A1 and VEGF-receptor 2 (VEGF-R2) are associated in a complex. Moreover, the presence of Sema6D promotes the tyrosine phosphorylation of VEGF-R2 in a plexin-A1-dependent manner. This is necessary for basal and Sema6D-induced NF-kappaB transcriptional activity, and NF-kappaB mediates tumor cell survival. Expression of Sema6D and plexin-A1 is induced by asbestos fibers and overexpression of plexin-A1 in nonmalignant mesothelial cells inhibits cell death after asbestos exposure. This work identifies a new biological function of semaphorins in cancer cells and suggests the involvement of an undescribed survival pathway during MPM tumorigenesis.

Enhanced Antitumor Therapy by Inhibition of p21waf1 in Human Malignant Mesothelioma

Purpose: The p21 cyclin-dependent kinase inhibitor was frequently expressed in human malignant pleural mesothelioma (MPM) tissues as well as cell lines. Recent data indicate that p21 keeps tumor cells alive after DNA damage, favoring a survival advantage. In this study, we assessed the possibility of p21 suppression as a therapeutic target for MPM. Experimental Design: We established two different MPM-derived (from H28 and H2052 cells) subclones using vector-based short hairpin RNA (shRNA). Then, chemosensitivity against low doses of antineoplastic DNA-damaging agents was investigated by colony formation assays, and furthermore, the type of cell response induced by these drugs was analyzed. To examine the effect of p21 shRNA on chemosensitivity in vivo, tumor formation assays in nude mice were done. Results: In colony formation assay, the IC50 of doxorubicin was 33 ± 3.0 nmol/L in p21 shRNA-transfected cells with respect to 125 ± 10 nmol/L of control vector–transfected cells. This enhancement of growth inhibition was achieved by converting a senescence-like growth arrest to apoptosis in response to doxorubicin, etoposide, and CPT11. In the in vivo assays, CPT11 and loss-of-expression of p21 in combination led to considerable suppression of tumor growth associated with a substantially enhanced apoptotic response, whereas CPT11 alone was ineffective at inducing these responses. Conclusions: These results indicated that p21 might play an important role in chemosensitivity to anticancer agents, and the suppression of its expression might be a potential therapeutic target for MPM.

5-Lipoxygenase antagonizes genotoxic stress-induced apoptosis by altering p53 nuclear trafficking

5‐Lipoxygenase (5‐LO) promotes cancer cell proliferation and survival by unclear mechanisms. Here, we show that 5‐LO expression and activity were induced by genotoxic agents in a p53‐independent manner and antagonized p53‐ or genotoxic drug‐induced apoptosis in a variety of cancer cells. 5‐LO inhibited p53‐governed transactivation of the pro‐apoptotic genes bax and pig3 but not of p21WAF1/CIP1 or mdm2. This may be explained by 5‐LO capability to inhibit the binding of p53 to promyelocytic leukemia protein (PML) and p53 subnuclear relocalization into PML‐nuclear bodies in response to genotoxic stress. Interestingly, 5‐LO activity appears to be involved in nuclear retention and inactivation of wild‐type p53 in malignant mesothelioma cells. In these cells, genetic or pharmacological inhibition of 5‐LO enabled suppression of in vitro tumorigenicity by low doses of chemotherapeutic drugs. Together, these results uncover novel functions of 5‐LO and contribute to the understanding of 5‐LO involvement in tumor progression. Moreover, they provide a rationale to the therapeutic use of 5‐LO inhibitors to enhance cancer chemosensitivity in selected tumors.