Ilaria Fuso Nerini

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.

Association between miR-200c and the survival of patients with stage I epithelial ovarian cancer: a retrospective study of two independent tumour tissue collections.

BACKGROUND International Federation of Gynecology and Obstetrics stage I epithelial ovarian cancer (EOC) has a significantly better prognosis than stage III/IV EOC, with about 80% of patients surviving at 5 years (compared with about 20% of those with stage III/IV EOC). However, 20% of patients with stage I EOC relapse within 5 years. It is therefore crucial that the biological properties of stage I EOCs are further elucidated. MicroRNAs (miRNAs) have shown diagnostic and prognostic potential in stage III and IV EOCs, but the small number of patients diagnosed with stage I EOC has so far prevented an investigation of its molecular features. We profiled miRNA expression in stage I EOC tumours to assess whether there is a miRNA signature associated with overall and progression-free survival (PFS) in stage I EOC. METHODS We analysed tumour samples from 144 patients (29 of whom relapsed) with stage I EOC gathered from two independent tumour tissue collections (A and B), both with a median follow-up of 9 years. 89 samples from tumour tissue collection A were stratified into a training set (51 samples, 15 of which were from patients who relapsed) for miRNA signature generation, and into a validation set (38 samples, seven of which were from patients who relapsed) for signature validation. Tumour tissue collection B (55 samples, seven of which were from patients who relapsed) was used as an independent test set. The Cox proportional hazards model and the log-rank test were used to assess the correlation of quantitative reverse transcription PCR (qRT-PCR)-validated miRNAs with overall survival and PFS. FINDINGS A signature of 34 miRNAs associated with survival was generated by microarray analysis in the training set. In both the training set and validation set, qRT-PCR analysis confirmed that 11 miRNAs (miR-214, miR-199a-3p, miR-199a-5p, miR-145, miR-200b, miR-30a, miR-30a*, miR-30d, miR-200c, miR-20a, and miR-143) were expressed differently in relapsers compared with non-relapsers. Three of these miRNAs (miR-200c, miR-199a-3p, miR-199a-5p) were associated with PFS, overall survival, or both in multivariate analysis. qRT-PCR analysis in the test set confirmed the downregulation of miR-200c in relapsers compared with non-relapsers, but not the upregulation of miR-199a-3p and miR-199a-5p. Multivariate analysis confirmed that downregulation of miR-200c in the test set was associated with overall survival (HR 0·094, 95% CI 0·012-0·766, p=0·0272) and PFS (0·035, 0·004-0·311; p=0·0026), independent of clinical covariates. INTERPRETATION miR-200c has potential as a predictor of survival, and is a biomarker of relapse, in stage I EOC. FUNDING Nerina and Mario Mattioli Foundation, Cariplo Foundation (Grant Number 2010-0744), and the Italian Association for Cancer Research.

Resistance to platinum-based chemotherapy is associated with epithelial to mesenchymal transition in epithelial ovarian cancer

BACKGROUND The present study is aimed to identify genetic pathways correlated with chemoresistance in epithelial ovarian cancer (EOC). METHODS We compared the molecular profiles of 23 tumour biopsies of stage III-IV (training set) at primary surgery, before chemotherapy, to the profile from the same patients at second surgery, after several lines of platinum (Pt)-based chemotherapy when the tumours were resistant. In the hypothesis that identified markers were related to Pt-resistance and to prognosis, we validated this signature in 52 EOC taken at primary surgery (validation set) selected to be either very sensitive to the first line therapy, i.e. not relapsing before one year from the end of therapy, or resistant, i.e. relapsing within 6 months from the end of therapy. RESULTS In the training set, we identified a resistance signature indicative of the activation of epithelial to mesenchymal transition (EMT) by transforming growth factor (TGF)-beta pathway. We then validated this signature in 52 EOC taken at primary surgery (validation set). Some genes involved in EMT, such as BMP and activin membrane-bound inhibitor (BAMBI), and mir-141 resulted in association with overall or progression free survival. CONCLUSION Some genes involved in EMT were associated to overall or progression free survival, suggesting EMT as vital to the resistance mechanisms.

miRNA Landscape in Stage I Epithelial Ovarian Cancer Defines the Histotype Specificities

Purpose: Epithelial ovarian cancer (EOC) is one of the most lethal gynecologic diseases, with survival rate virtually unchanged for the past 30 years. EOC comprises different histotypes with molecular and clinical heterogeneity, but up till now the present gold standard platinum-based treatment has been conducted without any patient stratification. The aim of the present study is to generate microRNA (miRNA) profiles characteristic of each stage I EOC histotype, to identify subtype-specific biomarkers to improve our understanding underlying the tumor mechanisms. Experimental Design: A collection of 257 snap-frozen stage I EOC tumor biopsies was gathered together from three tumor tissue collections and stratified into independent training (n = 183) and validation sets (n = 74). Microarray and quantitative real-time PCR (qRT-PCR) were used to generate and validate the histotype-specific markers. A novel dedicated resampling inferential strategy was developed and applied to identify the highest reproducible results. mRNA and miRNA profiles were integrated to identify novel regulatory circuits. Results: Robust miRNA markers for clear cell and mucinous histotypes were found. Specifically, the clear cell histotype is characterized by a five-fold (log scale) higher expression of miR-30a and miR-30a*, whereas mucinous histotype has five-fold (log scale) higher levels of miR-192/194. Furthermore, a mucinous-specific regulatory loop involving miR-192/194 cluster and a differential regulation of E2F3 in clear cell histotype were identified. Conclusions: Our findings showed that stage I EOC histotypes have their own characteristic miRNA expression and specific regulatory circuits. Clin Cancer Res; 19(15); 4114–23. ©2013 AACR.

Bevacizumab-induced inhibition of angiogenesis promotes a more homogeneous intratumoral distribution of paclitaxel, improving the antitumor response

The antitumor activity of angiogenesis inhibitors is reinforced in combination with chemotherapy. It is debated whether this potentiation is related to a better drug delivery to the tumor due to the antiangiogenic effects on tumor vessel phenotype and functionality. We addressed this question by combining bevacizumab with paclitaxel on A2780-1A9 ovarian carcinoma and HT-29 colon carcinoma transplanted ectopically in the subcutis of nude mice and on A2780-1A9 and IGROV1 ovarian carcinoma transplanted orthotopically in the bursa of the mouse ovary. Paclitaxel concentrations together with its distribution by MALDI mass spectrometry imaging (MALDI MSI) were measured to determine the drug in different areas of the tumor, which was immunostained to depict vessel morphology and tumor proliferation. Bevacizumab modified the vessel bed, assessed by CD31 staining and dynamic contrast enhanced MRI (DCE-MRI), and potentiated the antitumor activity of paclitaxel in all the models. Although tumor paclitaxel concentrations were lower after bevacizumab, the drug distributed more homogeneously, particularly in vascularized, non-necrotic areas, and was cleared more slowly than controls. This happened specifically in tumor tissue, as there was no change in paclitaxel pharmacokinetics or drug distribution in normal tissues. In addition, the drug concentration and distribution were not influenced by the site of tumor growth, as A2780-1A9 and IGROV1 growing in the ovary gave results similar to the tumor growing subcutaneously. We suggest that the changes in the tumor microenvironment architecture induced by bevacizumab, together with the better distribution of paclitaxel, may explain the significant antitumor potentiation by the combination. Mol Cancer Ther; 15(1); 125–35. ©2015 AACR.

Heterogeneity of paclitaxel distribution in different tumor models assessed by MALDI mass spectrometry imaging

The penetration of anticancer drugs in solid tumors is important to ensure the therapeutic effect, so methods are needed to understand drug distribution in different parts of the tumor. Mass spectrometry imaging (MSI) has great potential in this field to visualize drug distribution in organs and tumor tissues with good spatial resolution and superior specificity. We present an accurate and reproducible imaging method to investigate the variation of drug distribution in different parts of solid tumors. The method was applied to study the distribution of paclitaxel in three ovarian cancer models with different histopathological characteristics and in colon cancer (HCT116), breast cancer (MDA-MB-231) and malignant pleural mesothelioma (MPM487). The heterogeneous drug penetration in the tumors is evident from the MALDI imaging results and from the images analysis. The differences between the various models do not always relate to significant changes in drug content in tumor homogenate examined by classical HPLC analysis. The specificity of the method clarifies the heterogeneity of the drug distribution that is analyzed from a quantitative point of view too, highlighting how marked are the variations of paclitaxel amounts in different part of solid tumors.

Combination therapy in cancer: effects of angiogenesis inhibitors on drug pharmacokinetics and pharmacodynamics

Validated preclinical studies have provided evidence that anti-vascular endothelial growth factor (VEGF) compounds enhance the activity of subsequent antitumor therapy, but the mechanism of this potentiation is far from clear. The most widespread explanation is enhanced delivery of therapeutics due to vascular remodeling, lower interstitial pressure, and increased blood flow. While the antiangiogenic effects on vascular morphology have been fairly consistent in both preclinical and clinical settings, the improvement of tumor vessel function is debated. This review focuses on the effect of anti-VEGF therapy on tumor microenvironment morphology and functions, and its therapeutic benefits when combined with other therapies. The uptake and spatial distribution of chemotherapeutic agents into the tumor after anti-VEGF are examined.

A Systems Biology Approach to Characterize the Regulatory Networks Leading to Trabectedin Resistance in an In Vitro Model of Myxoid Liposarcoma

Trabectedin, a new antitumor compound originally derived from a marine tunicate, is clinically effective in soft tissue sarcoma. The drug has shown a high selectivity for myxoid liposarcoma, characterized by the translocation t(12;16)(q13; p11) leading to the expression of FUS-CHOP fusion gene. Trabectedin appears to act interfering with mechanisms of transcription regulation. In particular, the transactivating activity of FUS-CHOP was found to be impaired by trabectedin treatment. Even after prolonged response resistance occurs and thus it is important to elucidate the mechanisms of resistance to trabectedin. To this end we developed and characterized a myxoid liposarcoma cell line resistant to trabectedin (402-91/ET), obtained by exposing the parental 402-91 cell line to stepwise increases in drug concentration. The aim of this study was to compare mRNAs, miRNAs and proteins profiles of 402-91 and 402-91/ET cells through a systems biology approach. We identified 3,083 genes, 47 miRNAs and 336 proteins differentially expressed between 402-91 and 402-91/ET cell lines. Interestingly three miRNAs among those differentially expressed, miR-130a, miR-21 and miR-7, harbored CHOP binding sites in their promoter region. We used computational approaches to integrate the three regulatory layers and to generate a molecular map describing the altered circuits in sensitive and resistant cell lines. By combining transcriptomic and proteomic data, we reconstructed two different networks, i.e. apoptosis and cell cycle regulation, that could play a key role in modulating trabectedin resistance. This approach highlights the central role of genes such as CCDN1, RB1, E2F4, TNF, CDKN1C and ABL1 in both pre- and post-transcriptional regulatory network. The validation of these results in in vivo models might be clinically relevant to stratify myxoid liposarcoma patients with different sensitivity to trabectedin treatment.

Abstract 3377: Bevacizumab-improved distribution of paclitaxel in ovarian cancer xenografts potentiates antitumor efficacy

Angiogenesis inhibitors have shown antitumor efficacy when combined with chemotherapy. It has been proposed that this potentiation is related to better drug delivery to the tumor due to improved vessel functionality. This work combined classical pharmacokinetics and imaging analysis to study the delivery and distribution of paclitaxel after bevacizumab in ovarian cancer. The study was conducted on ovarian cancer models (A2780-1A9 and IGROV-1) xenografted orthotopically under the bursa of the nude mouse ovary. Mice bearing tumors in the ovary, treated with bevacizumab (150μg/mouse) and 24 hours later with paclitaxel (60mg/kg), were sacrificed and tumor excised for analysis, or monitored for anti-tumor activity. Paclitaxel spatial distribution after bevacizumab was analyzed by MALDI-Mass Spectrometry Imaging (MALDI-MSI) on frozen tumor slices, and compared with adjacent histochemical images stained for CD31 (vessel analysis) and Ki-67 (proliferation/necrosis); total paclitaxel in tumor homogenates, liver and plasma was evaluated by high-performance liquid chromatography (HPLC). Tumor growth of A2780-1A9 and IGROV-1, carrying the firefly luciferase gene luc2, was monitored by Bioluminescence imaging (BLI) and survival recorded. Bevacizumab decreased tumor vessel number/size compared to vehicle treated mice, but potentiated the antitumor activity of paclitaxel in both models. MALDI-MSI showed that after bevacizumab, paclitaxel was more homogenously distributed and mainly in actively proliferating areas of the tumor where vessels were uniformly diffused, despite paclitaxel concentration did not increase after antiangiogenic drug. No changes in paclitaxel systemic exposure was found in normal tissue. These findings suggest that a more uniform distribution of paclitaxel in the tumor of the mouse ovary is responsible for the better antitumor activity of the combination. A.D. and I.F.N. are the recipients of a fellowship from the Italian Foundation for Cancer Research (FIRC). Citation Format: Marta Cesca, Lavinia Morosi, Alexander Berndt, Ilaria Fuso Nerini, Alessandra Decio, Massimo Zucchetti, Raffaella Giavazzi. Bevacizumab-improved distribution of paclitaxel in ovarian cancer xenografts potentiates antitumor efficacy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3377.