Co-targeting the IGF system and HIF-1 inhibits migration and invasion by (triple-negative) breast cancer cells

BACKGROUND: Metastatic triple-negative breast cancer is mostly incurable, due to lack of suitable drug targets. The insulin-like growth factor (IGF) system could provide such a target, and IGF-1 receptor (IGF-1R)-directed agents are already available, but seem unable to control all the complexities of the system, including crosstalk with hypoxia-inducible pathways. METHODS: Migration of triple-negative MDA-231 breast cancer cells and its modulation by IGFs, the IGF-1R inhibitor NVP-AEW541 and the IGF-2-sequestering monoclonal antibody MAB292 were assessed by the scratch wound healing and Boyden chamber assays; the effect of topotecan (inhibiting hypoxia-inducible factor-1 (HIF-1)) under hypoxia was also evaluated. Constitutive as well as drug-modulated levels of components of the IGF and HIF-1 pathways were evaluated by western blotting and qPCR. RESULTS: IGF-induced migration of MDA-231 cells was not abrogated by the IGF-1R inhibitor NVP-AEW541, whereas IGF-2 sequestration by MAB292 significantly reduced cell migration. Under hypoxia, topotecan was also effective, likely by reducing HIF-1-induced IGF-2 release. Simultaneous targeting of IGF-1R and IGF-2 or HIF-1 completely abolished cell migration. CONCLUSIONS: IR activation may account for the failure of NVP-AEW541 to suppress MDA-231 cell migration. Ligand-targeting compounds, or co-inhibition of the IGF and HIF-1 systems, may prevent activation of compensatory signalling, thereby providing a valuable addition to IGF-1R inhibitor-based therapies

A systems biology approach to investigate the mechanism of action of trabectedin in a model of myelomonocytic leukemia

This study was designed to investigate the mode of action of trabectedin in myelomonocytic leukemia cells by applying systems biology approaches to mine gene expression profiling data and pharmacological assessment of the cellular effects. Significant enrichment was found in regulons of target genes inferred for specific transcription factors, among which MAFB was the most upregulated after treatment and was central in the transcriptional network likely to be relevant for the specific therapeutic effects of trabectedin against myelomonocytic cells. Using the Connectivity Map, similarity among transcriptional signatures elicited by treatment with different compounds was investigated, showing a high degree of similarity between transcriptional signatures of trabectedin and those of the topoisomerase I inhibitor, irinotecan, and an anti-dopaminergic antagonist, thioridazine. The study highlights the potential importance of systems biology approaches to generate new hypotheses that are experimentally testable to define the specificity of the mechanism of action of drugs.The Pharmacogenomics Journal advance online publication, 13 December 2016; doi:10.1038/tpj.2016.76

Antitumour activity of trabectedin in myelodysplastic/myeloproliferative neoplasms

Background: Juvenile myelomonocytic leukaemia (JMML) and chronic myelomonocytic leukaemia (CMML) are myelodysplastic myeloproliferative (MDS/MPN) neoplasms with unfavourable prognosis and without effective chemotherapy treatment. Trabectedin is a DNA minor groove binder acting as a modulator of transcription and interfering with DNA repair mechanisms; it causes selective depletion of cells of the myelomonocytic lineage. We hypothesised that trabectedin might have an antitumour effect on MDS/MPN. Methods: Malignant CD14+ monocytes and CD34+ haematopoietic progenitor cells were isolated from peripheral blood/bone marrow mononuclear cells. The inhibition of CFU-GM colonies and the apoptotic effect on CD14+ and CD34+ induced by trabectedin were evaluated. Trabectedin's effects were also investigated in vitro on THP-1, and in vitro and in vivo on MV-4-11 cell lines. Results: On CMML/JMML cells, obtained from 20 patients with CMML and 13 patients with JMML, trabectedin - at concentration pharmacologically reasonable, 1-5 nM - strongly induced apoptosis and inhibition of growth of haematopoietic progenitors (CFU-GM). In these leukaemic cells, trabectedin downregulated the expression of genes belonging to the Rho GTPases pathway (RAS superfamily) having a critical role in cell growth and cytoskeletal dynamics. Its selective activity on myelomonocytic malignant cells was confirmed also on in vitro THP-1 cell line and on in vitro and in vivo MV-4-11 cell line models. Conclusions: Trabectedin could be good candidate for clinical studies in JMML/CMML patients

n-3 Docosapentaenoic acid-derived protectin D1 promotes resolution of neuroinflammation and arrests epileptogenesis.

This is a pre-copyedited, author-produced version of an article accepted for publication in Brain: A Journal of Neurology following peer review. The version of record Frigerio, F., et al. (2018). "n-3 Docosapentaenoic acid-derived protectin D1 promotes resolution of neuroinflammation and arrests epileptogenesis." Brain: awy247-awy247. is available online at: xxxxxxx https://doi.org/10.1093/brain/awy247Epilepsy therapy is based on drugs that treat the symptoms rather than the underlying mechanisms of the disease (epileptogenesis). There are no treatments for preventing seizures or improving disease prognosis, including neurological comorbidities. The search of pathogenic mechanisms of epileptogenesis highlighted that neuroinflammatory cytokines [i.e. interleukin-1β (IL-1β), tumour necrosis factor-α (Tnf-α)] are induced in human and experimental epilepsies, and contribute to seizure generation in animal models. A major role in controlling the inflammatory response is played by specialized pro-resolving lipid mediators acting on specific G-protein coupled receptors. Of note, the role that these pathways have in epileptogenic tissue remains largely unexplored. Using a murine model of epilepsy, we show that specialized pro-resolving mechanisms are activated by status epilepticus before the onset of spontaneous seizures, but with a marked delay as compared to the neuroinflammatory response. This was assessed by measuring the time course of mRNA levels of 5-lipoxygenase (Alox5) and 15-lipoxygenase (Alox15), the key biosynthetic enzymes of pro-resolving lipid mediators, versus Il1b and Tnfa transcripts and proteins. In the same hippocampal tissue, we found a similar delayed expression of two main pro-resolving receptors, the lipoxin A4 receptor/formyl peptide receptor 2 and the chemerin receptor. These receptors were also induced in the human hippocampus after status epilepticus and in patients with temporal lobe epilepsy. This evidence supports the hypothesis that the neuroinflammatory response is sustained by a failure to engage pro-resolving mechanisms during epileptogenesis. Lipidomic LC-MS/MS analysis showed that lipid mediator levels apt to resolve the neuroinflammatory response were also significantly altered in the hippocampus during epileptogenesis with a shift in the biosynthesis of several pro-resolving mediator families including the n-3 docosapentaenoic acid (DPA)-derived protectin D1. Of note, intracerebroventricular injection of this mediator during epileptogenesis in mice dose-dependently reduced the hippocampal expression of both Il1b and Tnfa mRNAs. This effect was associated with marked improvement in mouse weight recovery and rescue of cognitive deficit in the novel object recognition test. Notably, the frequency of spontaneous seizures was drastically reduced by 2-fold on average and the average seizure duration was shortened by 40% after treatment discontinuation. As a result, the total time spent in seizures was reduced by 3-fold in mice treated with n-3 DPA-derived protectin D1. Taken together, the present findings demonstrate that epilepsy is characterized by an inadequate engagement of resolution pathways. Boosting endogenous resolution responses significantly improved disease outcomes, providing novel treatment avenues.F.F. was a recipient of a fellowship from UCB Biopharma. This work was supported by European Union’s Seventh Framework Programme (FP7/2007-2013) under grant agreement n. 602102 (EPITARGET to A.V., M.C.W., E.A.V., E.A.), Citizen United for Research in Epilepsy (CURE) (A.V.), the Epilepsy Research UK (M.C.W.), and in part by NIH grants RO1 NS086423 and RO1NS039587 (M.P.). This work was also supported by a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (Grant number: 107613/Z/15/Z), funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (Grant number: 677542), and the Barts Charity (Grant number: MGU0343). The Norwegian Research Council is acknowledged for funding to T.V.H. (FRIPROFRINATEK 230407)

The BH3-mimetic obatoclax reduces HIF-1\u3b1 levels and HIF-1 transcriptional activity and sensitizes hypoxic colon adenocarcinoma cells to 5-fluorouracil.

Activation of hypoxia-inducible factor (HIF)-1 is a feature of hypoxic solid tumors that has been associated with drug resistance, mainly due to disruption of Bcl-2 family dynamics. Resetting the balance in favor of proapoptotic family members is an attractive therapeutic goal that has been pursued by developing BH3-mimetic compounds. In the present study we evaluated the response of human colon adenocarcinoma cells to the BH3-mimetic obatoclax (OBX), in terms of growth arrest, apoptosis and autophagy, in the presence or absence of HIF-1\u3b1-stabilizing conditions; its possible effect on HIF-1\u3b1 expression and HIF-1 activity; and the possibility to improve the response of colon cancer cells to cytotoxic chemotherapeutics by combining them with OBX. Colon cancer cell response to the BH3-mimetic was unmodified by HIF-1 activation and OBX induced a decrease in HIF-1\u3b1 protein levels and HIF-1 transcriptional activity, probably by decreasing HIF-1\u3b1 synthesis and facilitating a VHL-independent proteasomal degradation pathway. Finally, a chemosensitizing effect of OBX with respect to 5-fluorouracil or oxaliplatin treatment was observed, highlighting the possibility that patients with hypoxic colon tumors might benefit from combined regimens including OBX

“miRNAs profile of bone marrow fibroblasts in multiple myeloma: relationship with disease progression and drug-resistance”

PURPOSE: microRNAs (miRs) regulate gene expression at post-transcriptional level modulating several biological processes. Bone marrow (BM) fibroblasts (or cancer associated fibroblasts, CAFs) from active multiple myeloma (MM) patients present an activated phenotype (FSP1+/FAP+/αSMA+), with higher proliferative rate compared to monoclonal gammopathy of undetermined significance (MGUS) CAFs1. BM CAFs from bortezomib (bort)-resistant patients are resistant in vitro to the drug and prevent bort-induced apoptosis of co-cultured MM cells2. Our purpose was to investigate whether a specific miR profile is associated to the phenotype and functional activities of BM CAFs in MGUS to MM transition and drug resistance. METHODS: miRs expression was analyzed by microarray and validated by qRT-PCR and flow cytometry on CAFs purified from BM aspirates of MGUS and MM patients. miRs target genes were identified by interrogating different tools commonly used to predict human miR gene targets and validated by western blot analysis. miRs functional effects were analyzed in CAFs transiently transfected with miRCURY LNA inhibitors and mimics. RESULTS AND DISCUSSION: MM and MGUS CAFs showed a different miRs profile, including 9 up-regulated and 17 down-regulated miRs. Among the over-expressed miRs, we focused on miRs showing a major significant p-value: miR-27b-3p and -214-3p. Target genes of miR-27b-3p and -214-3p were FBXW7 and PTEN, respectively, involved in cell apoptosis, proliferation and CAFs activation. Inhibition of miR-27b-3p induced the over-expression of FBXW7, an ubiquitin ligase, which negatively modulated the expression of MCL-1, NOTCH and Cyclin E1/2. miR-214-3p inhibition increased PTEN levels down-regulating the AKT/GSK3 pathway and Cyclin D1. Finally, co-cultures of MM cells with CAFs and bort treatment increased miRs expression. CONCLUSIONS: MGUS to MM transition and drug resistance is related to a specific miRs profile. Over-expression of miR-27b-3p and -214-3p induces cell proliferation and resistance to spontaneous and bort-induced apoptosis in MM CAFs. BIBLIOGRAPHY: 1. Frassanito MA et al. Leukemia 2014. 2. Frassanito MA et al. Leukemia 2016

Identification of high-grade serous ovarian cancer miRNA species associated with survival and drug response in patients receiving neoadjuvant chemotherapy: A retrospective longitudinal analysis using matched tumor biopsies

Background: Neoadjuvant chemotherapy (NACT) has been recognized as a reliable therapeutic strategy in patients with unresectable advanced epithelial ovarian cancer (EOC). The molecular events leading to platinum (Pt) response in NACT settings have hitherto not been explored. In the present work, longitudinal changes of miRNA expression profile were investigated to identify miRNA families with prognostic role in high-grade serous EOC patients who received the NACT regimen. Patients and methods: One hundred sixty-four matched tumor biopsies taken at initial laparoscopic evaluation and at interval-debulking surgery (IDS) after four courses of Pt-based therapy were selected from 82 stage IIIC-IV high-grade serous-EOC patients that were judged unsuitable for complete primary debulking and subjected the NACT protocol. miRNA profiling by microarray, real-time PCR and immuno-histochemical staining for Smad2 phosphorylation (P-Smad2) were used for data analysis. Results: Analysis revealed that 369 miRNAs were differentially expressed in matched biopsies (referred to as DEMs). DEMs were not scattered across the genome, but clustered into families: miR-199, let-7, miR-30, miR-181 and miR-29. Multivariate analysis showed that miR-199a-3p, miR-199a-5p, miR-181a-5p and let-7g-5p associated with overall and progression- free survival (P0.05). Kaplan-Meier curves plotting concomitant expression of P-Smad2 and miR-181a-5p show significant differences in PFS and OS compared with those depicting the expression of each biomarker alone (P>0.001). Conclusions: This study describes several miRNA families with a prognostic role in the NACT setting. It also confirms that concomitant analysis of P-Smad2 and miR-181a-5p in surgical samples may be capable of identifying those ovarian cancer patients with poor outcome and little chance of response to Pt-based NACT