Eugenio Erba

Role of Macrophage Targeting in the Antitumor Activity of Trabectedin

There is widespread interest in macrophages as a therapeutic target in cancer. Here, we demonstrate that trabectedin, a recently approved chemotherapeutic agent, induces rapid apoptosis exclusively in mononuclear phagocytes. In four mouse tumor models, trabectedin caused selective depletion of monocytes/macrophages in blood, spleens, and tumors, with an associated reduction of angiogenesis. By using trabectedin-resistant tumor cells and myeloid cell transfer or depletion experiments, we demonstrate that cytotoxicity on mononuclear phagocytes is a key component of its antitumor activity. Monocyte depletion, including tumor-associated macrophages, was observed in treated tumor patients. Trabectedin activates caspase-8-dependent apoptosis; selectivity for monocytes versus neutrophils and lymphocytes is due to differential expression of signaling and decoy TRAIL receptors. This unexpected property may be exploited in different therapeutic strategies.

Ecteinascidin-743 (ET-743), a natural marine compound, with a unique mechanism of action

The mode of action of Ecteinascidin-743 (ET-743), a marine tetrahydroisoquinoline alkaloid isolated from Ecteinascidia turbinata, which has shown very potent antitumour activity in preclinical systems and encouraging results in Phase I clinical trials was investigated at a cellular level. Both SW620 and LoVo human intestinal carcinoma cell lines exposed for 1 h to ET-743 progress through S phase more slowly than control cells and then accumulate in the G2M phase. The sensitivity to ET-743 of G1 synchronised cells was much higher than that of cells synchronised in S phase and even higher than that of cells synchronised in G2M. ET-743 concentrations up to four times higher than the IC(50) value caused no detectable DNA breaks or DNA-protein cross-links as assessed by alkaline elution techniques. ET-743 induced a significant increase in p53 levels in cell lines expressing wild-type (wt) (p53). However, the p53 status does not appear to be related to the ET-743 cytotoxic activity as demonstrated by comparing the drug sensitivity in p53 (-/-) or (+/+) mouse embryo fibroblasts and in A2780 ovarian cancer cells or the A2780/CX3 sub-line transfected with a dominant-negative mutant TP53. The cytotoxic potency of ET-743 was comparatively evaluated in CHO cell lines proficient or deficient in nucleotide excision repair (NER), and it was found that ET-743 was approximately 7-8 times less active in ERCC3/XPB and ERCC1-deficient cells than control cells. The findings that G1 phase cells are hypersensitive and that NER-deficient cells are resistant to ET-743 indicate that the mode of action of ET-743 is unique and different from that of other DNA-interacting drugs.

Anti-inflammatory properties of the novel antitumor agent yondelis (trabectedin): inhibition of macrophage differentiation and cytokine production

Yondelis (Trabectedin) is a novel antitumor agent of marine origin extracted from the tunicate Ecteinascidia turbinata. This original compound is active against several human tumors including sarcoma and ovarian and breast adenocarcinoma, as evidenced in phase II clinical trials in advanced multitreated patients. Yondelis is a DNA minor groove binder that blocks cell cycle and interferes with inducible gene transcription in a selective manner. In this study, we investigated the immunomodulatory properties of Yondelis on leukocytes. Human blood monocytes were highly susceptible in vitro to its cytotoxic effect and underwent apoptosis at pharmacologically relevant concentrations (5 nmol/L), whereas lymphocytes were up to 5-fold less sensitive. Macrophages differentiated in vitro with macrophage colony-stimulating factor and tumor-associated macrophages (TAM), isolated from patients with ovarian cancer, were also susceptible. At subcytotoxic concentrations, Yondelis inhibited the in vitro differentiation of monocytes to macrophages. In tumor-treated patients, drug infusion caused a selective decrease of monocyte counts and of ex vivo macrophage differentiation. The in vitro production of two proinflammatory mediators, CCL2 and IL-6, was markedly reduced by Yondelis in monocytes, macrophages, TAM, and freshly isolated ovarian tumor cells. The chemokine CCL2 is the major determinant of monocyte recruitment at tumor sites, whereas IL-6 is a growth factor for ovarian tumors. In view of the protumor activity of TAM and of the strong association between chronic inflammation and cancer progression, the inhibitory effect of Yondelis on macrophage viability, differentiation, and cytokine production is likely to contribute to the antitumor activity of this agent in inflammation-associated human tumors.

Antitumor and anti-inflammatory effects of trabectedin on human myxoid liposarcoma cells.

Inflammatory mediators present in the tumor milieu may promote cancer progression and are considered promising targets of novel biological therapies. We previously reported that the marine antitumor agent trabectedin, approved in Europe in 2007 for soft tissue sarcomas and in 2009 for ovarian cancer, was able to downmodulate the production of selected cytokines/chemokines in immune cells. Patients with myxoid liposarcoma (MLS), a subtype characterized by the expression of the oncogenic transcript FUS-CHOP, are highly responsive to trabectedin. The drug had marked antiproliferative effects on MLS cell lines at low nanomolar concentrations. We tested the hypothesis that trabectedin could also affect the inflammatory mediators produced by cancer cells. Here, we show that MLS express several cytokines, chemokines, and growth factors (CCL2, CCL3, CCL5, CXCL8, CXCL12, MIF, VEGF, SPARC) and the inflammatory and matrix-binder protein pentraxin 3 (PTX3), which build up a prominent inflammatory environment. In vitro treatment with noncytotoxic concentrations of trabectedin selectively inhibited the production of CCL2, CXCL8, IL-6, VEGF, and PTX3 by MLS primary tumor cultures and/or cell lines. A xenograft mouse model of human MLS showed marked reduction of CCL2, CXCL8, CD68+ infiltrating macrophages, CD31+ tumor vessels, and partial decrease of PTX3 after trabectedin treatment. Similar findings were observed in a patient tumor sample excised after several cycles of therapy, indicating that the results observed in vitro might have in vivo relevance. In conclusion, trabectedin has dual effects in liposarcoma: in addition to direct growth inhibition, it affects the tumor microenvironment by reducing the production of key inflammatory mediators.

Trabectedin (ET-743) promotes differentiation in myxoid liposarcoma tumors

Differentiation is a complex set of events that can be blocked by rearrangements of regulatory genes producing fusion proteins with altered properties. In the case of myxoid liposarcoma (MLS) tumors, the causative abnormality is a fusion between the CHOP transcription factor and the FUS or EWS genes. CHOP belongs to and is a negative regulator of the large CAAT/enhancer binding protein family whose α, β,and δ members are master genes of adipogenesis. Recent clinical data indicate a peculiar sensitivity of these tumors to the natural marine compound trabectedin. One hypothesis is that the activity of trabectedin is related to the inactivation of the FUS-CHOP oncogene. We find that trabectedin causes detachment of the FUS-CHOP chimera from targeted promoters. Reverse transcription-PCR and chromatin immunoprecipitation analysis in a MLS line and surgical specimens of MLS patients in vivo show activation of the CAAT/enhancer binding protein–mediated transcriptional program that leads to morphologic changes of terminal adipogenesis. The activity is observed in cells with type 1 but not type 8 fusions. Hence, the drug induces maturation of MLS lipoblasts in vivo by targeting the FUS-CHOP–mediated transcriptional block. These data provide a rationale for the specific activity of trabectedin and open the perspective of combinatorial treatments with drugs acting on lipogenic pathways. [Mol Cancer Ther 2009;8(2):449–57]

The combination of yondelis and cisplatin is synergistic against human tumor xenografts.

Yondelis (trabectidin, ET-743) is a marine natural product that has shown activity both in preclinical systems and in human malignancies such as soft tissue sarcoma and ovarian cancers that are resistant to previous chemotherapies. Molecular pharmacological studies indicated that Yondelis interacts with DNA and DNA repair systems in a way that is different from Cisplatin (DDP). The current study was designed to investigate the effects of the combination of Yondelis and DDP in human cancer cell lines and in xenografts derived from different tumours. The in vitro studies performed in human TE-671 rhabdomyosarcoma, Igrov-1 and 1A9 human ovarian carcinoma cell lines showed additive effects or slight synergism. Several human tumour xenografts, such as TE-671 rhabdomyosarcoma, SK-N-DX neuroblastoma, FADU head and neck, LX-1 non-small cell lung cancer (NSCLC), H-187 melanoma and SKOV HOC 8 ovarian carcinoma, showed an antitumour effect for the combination that was greater than that of each drug when given as a single agent. No consistent changes in the activity were observed if Yondelis and DDP were given 1 h apart in sequence or simultaneously. An orthotopically transplanted human ovarian cancer HOC 8 growing in the peritoneal cavity of nude mice was used that is insensitive to Yondelis alone and only moderately sensitive to DDP alone. The combination of the two drugs produced a dramatic increase of survival lasting several months. In conclusion, the combination of Yondelis and DDP is synergistic in vivo (i.e. the antitumour effect is greater than that of each drug used as a single agent at the maximum tolerated dose (MTD)) in different human tumour xenografts. The two drugs can be combined at the MTD of each drug, thus indicating there are no overlapping toxicities. These results provide a rationale for testing the combination of Yondelis and DDP in the clinic.

Role of homologous recombination in trabectedin-induced DNA damage

Trabectedin (ET-743, Yondelis) is a natural marine compound with antitumour activity currently undergoing phase II/III clinical trials. The mechanism of the drugs action is still to be defined, even though it has been clearly demonstrated the key role of Nucleotide Excision Repair (NER). To get further insights into the drugs mode of action, we studied the involvement of the DNA-double strand break repair (DNA-DSB) pathways: homologous and non-homologous recombination, both in budding yeasts and in mammalian cells and the possible cross-talk between NER and these repair pathways. Budding yeasts and mammalian cells deficient in the non-homologous end-joining pathway were moderately sensitive to trabectedin, while systems deficient in the homologous recombination pathway were extremely sensitive to the drug, with a 100-fold decrease in the IC50, suggesting that trabectedin-induced lesions are repaired by this pathway. The induction of Rad51 foci and the appearance of gamma-H2AX were chosen as putative markers for DNA-DSBs and were studied at different time points after trabectedin treatment in NER proficient and deficient systems. Both were clearly detected only in the presence of an active NER, suggesting that the DSBs are not directly caused by the drug, but are formed during the processing/repair of the drug- induced lesions.

Chk1, but not Chk2, is involved in the cellular response to DNA damaging agents: differential activity in cells expressing or not p53.

Mammalian Chk1 and Chk2 protein kinases are two important components of the G2DNA damage checkpoint. They are activated by upstream kinases (ataxia telangectasiamutated gene and ATM and Rad 3 related gene) and interfere with the activity of thecdc2/cyclinB1 complex, necessary for the G2-M transition, through the inactivation of thecdc25 phosphatases (cdc25A and cdc25C). To understand the role of Chk1 and Chk2 in thecellular response to different anticancer agents, we knocked down the expression of eachprotein or simultaneously of both proteins by using the small interfering RNA techniquein the HCT-116 colon carcinoma cell line and in its isogenic systems in which p53 and p21have been inactivated by targeted homologous recombination. We here show thatinhibition of Chk1 but not of Chk2 in p21-/- and p53-/- cells caused a greater abrogationof G2 block induced by ionizing radiation and cis-diamine-dichloroplatinum treatmentsand a greater sensitisation to the same treatments than in the parental cell line with p53and p21 wild type proteins. These data further emphasise the role of Chk1 as a moleculartarget to inhibit in tumors with a defect in the G1 checkpoint with the aim of increasingthe selectivity and specificity of anticancer drug treatments.

Mode of action of thiocoraline. a natural marine compound with anti-tumour activity

SummaryThiocoraline, a new anticancer agent derived from the marine actinomycete Micromonospora marina, was found to induce profound perturbations of the cell cycle. On both LoVo and SW620 human colon cancer cell lines, thiocoraline caused an arrest in G1 phase of the cell cycle and a decrease in the rate of S phase progression towards G2/M phases, as assessed by using bromodeoxyuridine/DNA biparametric flow cytometric analysis. Thiocoraline does not inhibit DNA-topoisomerase II enzymes in vitro, nor does it induce DNA breakage in cells exposed to effective drug concentrations. The cell cycle effects observed after exposure to thiocoraline appear related to the inhibition of DNA replication. By using a primer extension assay it was found that thiocoraline inhibited DNA elongation by DNA polymerase α at concentrations that inhibited cell cycle progression and clonogenicity. These studies indicate that the new anticancer drug thiocoraline probably acts by inhibiting DNA polymerase α activity.

The isothiocyanate produced from glucomoringin inhibits NF-kB and reduces myeloma growth in nude mice in vivo.

Glucosinolates (GLs), natural compounds extracted from Brassicaceae and precursors of isothiocyanates (ITCs), have been studied in the last decades mostly due to their chemopreventive activity and, more recently, for their potential use as novel chemotherapeutics. The aim of the present study was to investigate the in vitro and in vivo activity of glucomoringin (GMG), an uncommon member of the GLs family, and to compare it with glucoraphanin (GRA), one of the most studied GL. We have evaluated the potency of both compounds in inducing cell death, cell cycle perturbations, apoptosis, NF-kB inhibition and GST-pi activity in human carcinoma cells with different GST-pi contents as well as in human multiple myeloma and leukaemia cell lines. GMG-derived ITC (GMG-ITC) showed to be more effective compared to GRA-derived ITC (Sulforaphane), especially in inhibiting NF-kB activity and inducing apoptosis through a caspase-dependent pathway; these effects were more pronounced in myeloma cells, in which we could also observe a long lasting growth inhibitory effect, probably due to NF-kB inhibition, which is considered essential for myeloma cell survival. Both GLs were able to induce cell death in the muM range in all tested cell lines but caused cell cycle perturbations only in myeloma cells; they were also able to modulate the GST/GSH pathway by causing a 3-fold increase in GST-pi activity in MCF7 cells. In vivo study showed that pure GMG-ITC was only slightly active in a carcinoma mice model, whereas it had significant antitumoral activity in a myeloma model, causing little toxicity.