Supplementary MaterialsSupplementary figures and dining tables

Supplementary MaterialsSupplementary figures and dining tables. The influence Rabbit Polyclonal to Acetyl-CoA Carboxylase of the long non-coding RNA, EGFR-AS1, on ERLO efficacy was also addressed. Methods: The effect of BVZ/IFN/ERLO was tested on the growth of experimental tumors in nude mice. The presence of germline mutation in the EGFR was evaluated on cell lines and primary RCC cells. translation and transfections of expression vectors coding the wild-type or the EGFR mutated gene in HEK-293 cells were used to test the role of EGFR mutation of the ERLO efficacy. Correlation between EGFR/EGFR-AS1 expression and survival was analyzed with an online available data base (TCGA). Results: Tumor growth was strongly reduced by LUF6000 the triple combination BVZ/IFN/ERLO and linked to reduced levels of pro-angiogenic/pro-inflammatory cytokines of the ELR+CXCL family and to subsequent inhibition of vascularization, a decreased number of lymphatic vessels and polarization of macrophages towards the M1 phenotype. Cells isolated from surgical resection of human tumors presented a range of sensitivity to ERLO depending on the presence of a newly detected mutation in the EGFR and to the presence of EGFR-AS1. Conclusions: Our results point-out that this BVZ/IFN/ERLO combination deserves testing for the treatment of mRCC that have a specific mutation in the EGFR. Introduction Before the development of anti-angiogenic therapies (AAT), the outcome of mRCC was poor. The first treatment approved for mRCC was the humanized monoclonal antibody bevacizumab/Avastin (BVZ) in combination with the standard treatment interferon alpha (IFN), the only treatment that showed a modest efficacy 1. These drugs are aimed at asphyxiating the tumors, so they should be curative but the results of pivotal clinical trials were disappointing and gave only an increase in the time to progression and in the quality LUF6000 of life without a major improvement in overall survival 2, 3. The reasons for this poor efficacy depend on compensative mechanisms that allow tumor cells to escape drug-mediated cell death. Acquisition of dependence on alternative signaling pathways favoring cell proliferation and invasion has been described including the c-MET 4 as well as the neuropilin (NRP1/NRP2) 5, 6 pathways. Myeloid cells have already been mixed up in refractoriness to AAT 7 also. The current presence of redundant pro-angiogenic elements can be among the factors behind relapse to remedies concentrating on the VEGF/VEGFR pathway specifically the ELR+CXCL pro-angiogenic/pro-inflammatory cytokines 8, 9. Id of markers of response to treatment is an important challenge and may favor the discovery of new potent therapeutic targets 10, 11. The epidermal growth factor receptor (EGFR) is usually over-expressed in mRCC probably via EGR-1 dependent activation of its promoter 12. The hypoxia-inducible factors 1, 2 (HIF-1, 2) are constitutively active in the majority of mRCC because of frequent loss of function of the von Hippel-Lindau gene that stimulates the expression of the transforming growth factor (TGF- ), an activator of the EGFR pathway 13. Our previous results showed that this pressure of selection exerted by BVZ induced down-regulation of the phospho tyrosine phosphatase receptor kappa (PTPR), a natural inhibitor of EGFR activity resulting in the acquisition of increased proliferation of tumor cells 9. These cells were driven by over-activation of EGFR as attested by the level of phosphorylation and of the subsequent activation of the ERK/MAP kinase and PI3 kinase/AKT pathways. = 10). Statistical differences to the untreated mice are shown: *p 0.05; *** p 0.001. (B) Same experiment as described in a but using A498 cells. * p 0.05; ** p 0.01; *** p 0.001. * p 0.05; *** p 0.001. (C) Images of the 786-O tumors at the end of the experiments. (D) Images of A498 tumors at the end of the experiment. BVZ/IFN/ERLO strongly reduced tumor vessel density and prevented the development of lymphatic vessels LUF6000 We showed previously that BVZ alone stimulated experimental tumor growth. This unexpected result correlated with tumor vessel normalization and the development of a lymphatic network shown in the literature to be involved in tumor cell dissemination 9, 24. Considering these observations, we hypothesized that this triple combination may eradicate blood vessels and may prevent the development the lymphatics. The number of blood vessels decreased for 786-O tumors treated with BVZ/IFN.