Roberta Frapolli

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.

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.

G-Quadruplex Ligand RHPS4 Potentiates the Antitumor Activity of Camptothecins in Preclinical Models of Solid Tumors

Purpose: The formation of G-quadruplex structures at telomeric DNA sequences blocks telomerase activity, offering an original strategy to design and develop new antitumor agents. The pentacyclic acridinium salt RHPS4 is one of the most effective and selective G4 ligands able to rapidly disrupt telomere architecture, resulting in apoptosis of cancer cells. Here, we studied the therapeutic index of RHPS4 and its integration with chemotherapeutics in preclinical model of solid tumors. Experimental Design: The antitumoral activity of RHPS4 was evaluated on human xenografts of different histotypes and compared with that of standard antineoplastic agents. Moreover, the effect of RHPS4/chemotherapeutics combinations on cell survival was studied and the most favorable combination was evaluated on tumor-bearing mice. Results: RHPS4 was active in vivo as single agent and showed a high therapeutic efficacy when compared with conventional drugs. Moreover, RHPS4 had antitumoral activity in human melanoma xenografts inherently resistant to chemotherapy and exhibited antimetastatic activity. RHPS4 also showed a strong synergistic interaction with camptothecins and this effect was strictly dependent on the drug sequence employed. Treatment of mice with irinotecan followed by RHPS4 was able to inhibit and delay tumor growth and to increase mice survival. Conclusions: Our data show that RHPS4 has a good pharmacodynamic profile and in combination therapy produces a strong antitumoral activity, identifying this drug as promising agent for clinical development.

Mode of action of trabectedin in myxoid liposarcomas

To elucidate the mechanisms behind the high sensitivity of myxoid/round cell liposarcoma (MRCL) to trabectedin and the suggested selectivity for specific subtypes, we have developed and characterized three MRCL xenografts, namely ML017, ML015 and ML004 differing for the break point of the fusion gene FUS-CHOP, respectively of type I, II and III. FUS-CHOP binding to the promoters of some target genes such as Pentraxin 3 or Fibronectin 1, assessed by chromatin immunoprecipitation, was strongly reduced in the tumor 24 h after the first or the third weekly dose of trabectedin, indicating that the drug at therapeutic doses causes a detachment of the FUS-CHOP chimera from its target promoters as previously shown in vitro. Moreover, the higher sensitivity of MRCL types I and II appears to be related to a more prolonged block of the transactivating activity of the fusion protein. Doxorubicin did not affect the binding of FUS-CHOP to target promoters. Histologically, the response to trabectedin in ML017 and ML015 was associated with a marked depletion of non-lipogenic tumoral cells and vascular component, as well as lipidic maturation as confirmed by PPARγ2 expression in western Blot. By contrast, in ML004 no major changes either in the cellularity or in the amount of mature were found, and consistently PPARγ2 was null. In conclusion, the data support the view that the selective mechanism of action of trabectedin in MRCL is specific and related to its ability to cause a functional inactivation of the oncogenic chimera with consequent derepression of the adypocytic differentiation.

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.

Chemical characterization of Iraqi propolis samples and assessing their antioxidant potentials.

Propolis samples, collected from different geographical locations in Iraq (Baghdad, Dahuk, Mosul and Salah ad-Din), were analyzed and assessed for their anti-oxidant activity. Concentrations of phenolic compounds (flavonoids, phenolic acids and their esters) in propolis were estimated using high performance liquid chromatography coupled to electrospray mass spectrometry. Thirty-eight different compounds were identified and 33 of them were polyphenols. Other compounds were tentatively identified as clerodane diterpenoids, and one was considered unknown. Semi-quantitative measurements showed that phenolic acids and their esters were the predominant constituents in propolis extracts, followed by flavones and flavonols, and then flavanones and dihydroflavonols. Propolis samples were further spectrophotometrically characterized using the Folin-Ciocalteu reagent for the determination of total phenolic compounds. The free radical scavenging activities of propolis samples were also evaluated by using the 2,2-diphenyl-1-picrylhydrazyl assay. The results revealed that propolis extracts exhibited strong free radical scavenging activity.

Sequence dependent antitumour efficacy of the vascular disrupting agent ZD6126 in combination with paclitaxel

The clinical success of small-molecule vascular disrupting agents (VDAs) depends on their combination with conventional therapies. Scheduling and sequencing remain key issues in the design of VDA–chemotherapy combination treatments. This study examined the antitumour activity of ZD6126, a microtubule destabilising VDA, in combination with paclitaxel (PTX), a microtubule-stabilising cytotoxic drug, and the influence of schedule and sequence on the efficacy of the combination. Nude mice bearing MDA-MB-435 xenografts received weekly cycles of ZD6126 (200 mg kg−1 i.p.) administered at different times before or after PTX (10, 20, and 40 mg kg−1 i.v.). ZD6126 given 2 or 24 h after PTX showed no significant benefit, a result that was attributed to a protective effect of PTX against ZD6126-induced vascular damage and tumour necrosis, a hallmark of VDA activity. Paclitaxel counteracting activity was reduced by distancing drug administrations, and ZD6126 given 72 h after PTX potentiated the VDAs antitumour activity. Schedules with ZD6126 given before PTX improved therapeutic activity, which was paralleled by a VDA-induced increase in cell proliferation in the viable tumour tissue. Paclitaxel given 72 h after ZD6126 yielded the best response (50% tumours regressing). A single treatment with ZD6126 followed by weekly administration of PTX was sufficient to achieve a similar response (57% remissions). These findings show that schedule, sequence and timing are crucial in determining the antitumour efficacy of PTX in combination with ZD6126. Induction of tumour necrosis and increased proliferation in the remaining viable tumour tissue could be exploited as readouts to optimise schedules and maximise therapeutic efficacy.

Reduced Expression of the ROCK Inhibitor Rnd3 Is Associated with Increased Invasiveness and Metastatic Potential in Mesenchymal Tumor Cells

Background Mesenchymal and amoeboid movements are two important mechanisms adopted by cancer cells to invade the surrounding environment. Mesenchymal movement depends on extracellular matrix protease activity, amoeboid movement on the RhoA-dependent kinase ROCK. Cancer cells can switch from one mechanism to the other in response to different stimuli, limiting the efficacy of antimetastatic therapies. Methodology and Principal Findings We investigated the acquisition and molecular regulation of the invasion capacity of neoplastically transformed human fibroblasts, which were able to induce sarcomas and metastases when injected into immunocompromised mice. We found that neoplastic transformation was associated with a change in cell morphology (from fibroblastic to polygonal), a reorganization of the actin cytoskeleton, a decrease in the expression of several matrix metalloproteases and increases in cell motility and invasiveness. In a three-dimensional environment, sarcomagenic cells showed a spherical morphology with cortical actin rings, suggesting a switch from mesenchymal to amoeboid movement. Accordingly, cell invasion decreased after treatment with the ROCK inhibitor Y27632, but not with the matrix protease inhibitor Ro 28-2653. The increased invasiveness of tumorigenic cells was associated with reduced expression of Rnd3 (also known as RhoE), a cellular inhibitor of ROCK. Indeed, ectopic Rnd3 expression reduced their invasive ability in vitro and their metastatic potential in vivo. Conclusions These results indicate that, during neoplastic transformation, cells of mesenchymal origin can switch from a mesenchymal mode of movement to an amoeboid one. In addition, they point to Rnd3 as a possible regulator of mesenchymal tumor cell invasion and to ROCK as a potential therapeutic target for sarcomas.

Inactivation of DNA repair triggers neoantigen generation and impairs tumour growth

Molecular alterations in genes involved in DNA mismatch repair (MMR) promote cancer initiation and foster tumour progression. Cancers deficient in MMR frequently show favourable prognosis and indolent progression. The functional basis of the clinical outcome of patients with tumours that are deficient in MMR is not clear. Here we genetically inactivate MutL homologue 1 (MLH1) in colorectal, breast and pancreatic mouse cancer cells. The growth of MMR-deficient cells was comparable to their proficient counterparts in vitro and on transplantation in immunocompromised mice. By contrast, MMR-deficient cancer cells grew poorly when transplanted in syngeneic mice. The inactivation of MMR increased the mutational burden and led to dynamic mutational profiles, which resulted in the persistent renewal of neoantigens in vitro and in vivo, whereas MMR-proficient cells exhibited stable mutational load and neoantigen profiles over time. Immune surveillance improved when cancer cells, in which MLH1 had been inactivated, accumulated neoantigens for several generations. When restricted to a clonal population, the dynamic generation of neoantigens driven by MMR further increased immune surveillance. Inactivation of MMR, driven by acquired resistance to the clinical agent temozolomide, increased mutational load, promoted continuous renewal of neoantigens in human colorectal cancers and triggered immune surveillance in mouse models. These results suggest that targeting DNA repair processes can increase the burden of neoantigens in tumour cells; this has the potential to be exploited in therapeutic approaches.

Comparison of in vitro and in vivo biological effects of trabectedin, lurbinectedin (PM01183) and Zalypsis® (PM00104).

This study: (i) investigated the in vitro cytotoxicity and mode of action of lurbinectedin (PM01183) and Zalypsis® (PM00104) compared with trabectedin in cell lines deficient in specific mechanisms of repair, (ii) evaluated their in vivo antitumor activity against a series of murine tumors and human xenografts. The antiproliferative activity, the DNA damage and the cell cycle perturbations induced by the three compounds on tumor lines were very similar. Nucleotide Excision Repair (NER) deficient cells were approximately fourfold more resistant to trabectedin, lurbinectedin and Zalypsis®. Cells deficient in non‐homologous end joining (NHEJ), MRN complex and translesion synthesis (TLS) were slightly more sensitive to the three compounds (approximately fivefold) while cells deficient in homologous recombination (HR) were markedly more sensitive (150–200‐fold). All three compounds showed a good antitumor activity in several in vivo models. Lurbinectedin and trabectedin had a similar pattern of antitumor activity in murine tumors and in xenografts, whereas Zalypsis® appeared to have a distinct spectrum of activity. The fact that no relationship whatsoever was found between the in vitro cytotoxic potency and the in vivo antitumor activity, suggests that in addition to direct cytotoxic mechanisms other host‐mediated effects are involved in the in vivo pharmacological effects.