◥Progression on therapy in non-small cell lung carcinoma (NSCLC) is often evaluated radiographically, however, imagebased evaluation of said therapies may not distinguish disease progression due to intrinsic tumor drug resistance or inefficient tumor penetration of the drugs. Here we report that the inhibition of mutated EGFR promotes the secretion of a potent vasoconstrictor, endothelin-1 (EDN1), which continues to increase as the cells become resistant with a mesenchymal phenotype. As EDN1 and its receptor (EDNR) is linked to cancer progression, EDNRantagonists have been evaluated in several clinical trials with disappointing results. These trials were based on a hypothesis that the EDN1-EDNR axis activates the MAPK-ERK signaling pathway that is vital to the cancer cell survival; the trials were not designed to evaluate the impact of tumor-derived EDN1 in modifying tumor microenvironment or contributing to drug resistance. Ectopic overexpression of EDN1 in cells with mutated EGFR resulted in poor drug delivery and retarded growth in vivo but not in vitro. Intratumoral injection of recombinant EDN significantly reduced blood flow and subsequent gefitinib accumulation in xenografted EGFRmutant tumors. Furthermore, depletion of EDN1 or the use of endothelin receptor inhibitors bosentan and ambrisentan improved drug penetration into tumors and restored blood flow in tumorassociated vasculature. Correlatively, these results describe a simplistic endogenous yet previously unrealized resistance mechanism inherent to a subset of EGFR-mutant NSCLC to attenuate tyrosine kinase inhibitor delivery to the tumors by limiting drug-carrying blood flow and the drug concentration in tumors.Significance: EDNR antagonists can be repurposed to improve drug delivery in VEGFA-secreting tumors, which normally respond to TKI treatment by secreting EDN1, promoting vasoconstriction, and limiting blood and drug delivery.
<p>Supplementary Materials and Methods</p>
Advanced malignant melanoma is one of the most lethal cancers, because it is highly metastatic and refractory to conventional chemotherapy. About 60% of melanomas harbor oncogenic BRAF mutations which aberrantly activate MEK/ERK signaling pathway. BRAF and MEK inhibitors have been shown efficacious in patients with BRAF-mutant melanoma, but there is not effective target therapy for BRAF wild type melanomas. Unfortunately acquired resistance to BRAF targeted therapies is a common event: 50% of treated patients progressed within 6 to 7 months after the initiation of treatment. Resistance is associated with reactivation of the MAPK pathway (through development of de novo NRAS, NF1 or MEK mutations) or activation of parallel pathways, such as the PI3K/AKT pathway. Cancer stem cells (CSCs) represent a subpopulation of tumor cells that possess self-renewal and tumor initiation capacity. It has been postulated that CSC could be linked to acquired resistance to both conventional and targeted therapies. Salinomycin, an ionophore antibiotic, has been shown to selectively kill CSCs in several types of human cancers, most likely by interfering with ABC drug transporters, the Wnt/β-catenin signaling pathway, and other CSC pathways. In this study, we have analyzed a wide panel of human melanoma cell lines with a known mutational status of BRAF, tumor suppressors, and other frequently mutated oncogenes in melanoma, and their sensibility to several protein kinase inhibitors (PKIs) in vitro, including the BRAF inhibitor vemurafenib. We have combined these PKIs with the CSC inhibitor, salinomycin, in order to find therapeutically relevant synergies. To better characterize CSC phenotype, we carried out tumorsphere formation assays in vitro, FACS analysis of CSC canonical markers, to find that salinomycin sensitizes BRAF-wild type melanoma cell lines to the cytotoxic effect of particular PKIs. Importantly, we demonstrated that salinomycin-PKI combination therapy supresses the emergence of vemurafenib-resistant subpopulations in BRAF-mutant melanoma cell lines. Overall, we find that targeting CSC property of wild-type BRAF inhibitor with salinomycin significantly potentiates therapeutic activity of PKIs. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A292. Citation Format: Daniel Crespo, Ines Pulido, Maria L. Rodriguez, Salvador Aparisi, Alejandro Lopez, Margaret Soucheray, Fatima Al-Shahrour, Takeshi Shimamura, Angel Ortega, Julian Carretero. Salinomycin, an anti-cancer stem cell antibiotic, overcomes acquired resistance to BRAF inhibitors in BRAF-mutant human melanoma cell lines. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A292.
Despite the availability of the effective targeted therapies in lung cancer, such as EGFR tyrosine kinase inhibitors (TKIs), drug tolerance and acquired resistance are two common problems that negatively impact lung cancer patient survival. Consequently it is important to understand the molecular basis of the drug tolerance and resistance so that we could formulate effective strategies to ameliorate the efficacy of existing drug and to suppress the emergence of drug resistance. A burgeoning body of literature demonstrated that epigenetic changes by the methylation of DNA and histones are critical in acquired drug resistance, especially in those cancer cells with stem cell-like properties and epithelial to mesenchymal (EMT)-like features. EMT involves multiple and complex changes in the epigenome and therefore in the transcriptome, the proteome and the metabolome. However, in the context of NSCLC, there are no comprehensive studies linking epigenomic and metabolic reprogramming during EMT and the acquisition of drug resistance. One-carbon metabolic pathway is composed of the folate and methionine cycle, generating S-adenosylmethionine (SAM), which is the universal methyl donor for methylation reactions, including histone and DNA methylation. Interestingly, it has been demonstrated that the overexpression of specific methyl-transferases, such as NNMT, which catalyzes the transfer of the methyl group from SAM to nicotinamide, can impact the methylation potential of cancer cells, draining methyl groups from the methionine cycle and thereby decreasing protein and DNA methylation. Strikingly, NNMT depletion has been shown to restore methylation potential and attenuate the aggressive nature of several cancer cell lines with NNMT overexpression. However, its contribution to lung cancer progression and the drug resistance is still unknown. In this work we have discovered that NNMT is a poor prognosis factor and is significantly correlated with mesenchymal-like phenotypes in publicly available NSCLC clinical databases. We have generated multiple EGFR-TKI acquired resistant NSCLC models with EMT phenotype and utilized a multi-omic approach on the clinically-relevant resistant cell lines to identified that NNMT overexpression affects the nicotinamide pathway leading to deficient nicotinamide adenine dinucleotide (NAD) synthesis. This new cancer vulnerability of TKI-resistant, mesenchymal-like NSCLC cells is druggable using NAD depleting agents such as daporinad, a NAMPT inhibitor in phase II clinical trials for the treatment of B-cell chronic lymphocytic leukemia, cutaneous T-cell lymphoma and melanoma. Additionally, the quantitative measurement of the metabolite signature of the nicotinamide pathway in liquid biopsies may correlate with poor prognosis of TKI-treated patients, and emerges as a potential biomarker of TKI response and tumor progression in NSCLC patients. Based on these evidences and tools, we propose that targeting nicotinamide pathway may serve as a therapeutic tool for sensitizing drug-resistant NSCLC cells with an EMT phenotype to currently available treatments. Citation Format: Ines Pulido, Jeff Becker, Miguel Aupi, Juan Carlos García Cañaveras, Javier Alcacer, María Rodríguez, Paloma Martín, Javier Perales, Salvador Aparisi, Lourdes Chulia, Arantxa Lafuente, Maribel Alcoriza, Salvador Mena, Javier Pereda, José Galbis-Carabajal, Amelia Insa, Oscar Juan, Fatima Al-Shahrour, Manuel Sanchez del Pino, Agustín Lahoz, Takeshi Shimamura, Julian Carretero. Metabolic vulnerabilities of mesenchymal-like EGFR-mutant NSCLC cells with acquired resistance to tyrosine kinase inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-099.
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