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Autophagy inhibition improves the cytotoxic effects of receptor tyrosine kinase inhibitors.

ABSTRACT: Background:A growing field of evidence suggests the involvement of oncogenic receptor tyrosine kinases (RTKs) in cell transformation. Deregulated activity of RTKs in tumors can determine disease progression and therapeutic responses in several types of cancer, including neuroblastoma (NB). Therefore, RTKs targeting is a worthwhile challenge for the oncologists. Nevertheless, acquired resistance to RTK inhibitors (RTKi) remains a serious problem. Autophagy activation is among the possible obstacles for good efficacy of the therapy with RTKi. Methods:Under different treatment conditions we measured autophagic flux using immunoblot and immunofluorescence assays. Death induction was validated by trypan blue exclusion assay and FACS analysis (calcein-AM/propidium iodide). The NB cell lines SH-SY5Y and Kelly were used for the in vitro study. Results:In order to define whether autophagy might be a limiting factor for the efficacy of RTKi in NB cells, we firstly checked its activation following the treatment with several RTKi. Next, we investigated the possibility to increase their therapeutic efficiency by combining RTKi with autophagy blocking agents in vitro. We exploited the effectiveness of three RTKi either alone or in combination with autophagy inhibitors (Chloroquine-CQ and Spautin-1). We demonstrated that autophagy induction was drug-dependent, and that its inhibition increased the anti-tumor activity of a single RTKi unevenly. We observed that the combined use of blocking agents which impair late autophagy events, such as CQ, and RTKi can be more effective with respect to the use of RTKi alone. Conclusions:In the present report, we assessed the conditions under which autophagy is activated during the use of different RTKi currently in the pre-clinical evaluation for NB. We summarized the achievements of combined RTK/autophagy inhibitors treatment as a promising approach to enhance the efficacy of RTKi in impairing tumor cells viability.

SUBMITTER: Aveic S

PROVIDER: S-EPMC5916832 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Autophagy inhibition improves the cytotoxic effects of receptor tyrosine kinase inhibitors.

Aveic Sanja S Pantile Marcella M Polo Pierfrancesco P Sidarovich Viktoryia V De Mariano Marilena M Quattrone Alessandro A Longo Luca L Tonini Gian Paolo GP

Cancer cell international 20180424

<h4>Background</h4>A growing field of evidence suggests the involvement of oncogenic receptor tyrosine kinases (RTKs) in cell transformation. Deregulated activity of RTKs in tumors can determine disease progression and therapeutic responses in several types of cancer, including neuroblastoma (NB). Therefore, RTKs targeting is a worthwhile challenge for the oncologists. Nevertheless, acquired resistance to RTK inhibitors (RTKi) remains a serious problem. Autophagy activation is among the possible ...[more]

PMID: 29713246

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Project description:Despite the potential therapeutic efficacy of epithelial growth factor receptor (EGFR) inhibitors in the treatment of advanced stage bladder cancer, there currently is no clear evidence to support this hypothesis. In this study, we investigate whether the concurrent treatment of autophagy-blocking agents with EGFR inhibitors exerts synergistic anti-cancer effects in T24 and J82 human bladder cancer cells. Lapatinib and gefitinib were used as EGFR inhibitors, and bafilomycin A1 (BFA1), chloroquine (CQ) and 3-methyladenine (3-MA) were used as the pharmacologic inhibitors of autophagy activities. To assess the proliferative and self-renewal capabilities, the Cell Counting Kit-8 (CCK-8) assay and a clonogenic assay were performed, respectively. To examine apoptotic cell death, flow cytometry using annexin-V/propidium iodide (PI) was used. To measure the autophagy activities, the expression levels of LC3I and II was determined by Western blot analysis. To validate the synergistic effects of autophagy inhibition with EGFR inhibitors, we specifically blocked key autophagy regulatory gene ATG12 by transfection of small interference RNA and examined the phenotypic changes. Of note, lapatinib and gefitinib triggered autophagy activities in T24 and J82 human bladder cancer cells, as indicated by upregulation of LC3II. More importantly, inhibiting autophagy activities with pharmacologic inhibitors (BFA1, CQ or 3-MA) remarkably reduced the cell viabilities and clonal proliferation of T24 and J82 cells, compared to those treated with either of the agents alone. We also obtained similar results of the enhanced anti-cancer effects of EGFR inhibitors by suppressing the expression of ATG12. Notably, the apoptotic assay showed that synergistic anti-cancer effects were induced via the increase of apoptotic cell death. In summary, concomitant inhibition of autophagy activities potentiated the anti-cancer effects of EGFR inhibitors in human bladder cancer cells, indicating a novel therapeutic strategy to treat advanced bladder cancer.

Project description:Asparagine (N)-linked glycosylation is a posttranslational modification essential for the function of complex transmembrane proteins. However, targeting glycosylation for cancer therapy has not been feasible due to generalized effects on all glycoproteins. Here, we perform sensitivity screening of 94 lung cancer cell lines using NGI-1, a small-molecule inhibitor of the oligosaccharyltransferase (OST) that partially disrupts N-linked glycosylation, and demonstrate a selective loss of tumor cell viability. This screen revealed NGI-1 sensitivity in just 11 of 94 (12%) cell lines, with a significant correlation between OST and EGFR inhibitors. In EGFR-mutant non-small cell lung cancer with EGFR tyrosine kinase inhibitor (TKI) resistance (PC9-GR, HCC827-GR, and H1975-OR), OST inhibition maintained its ability to induce cell-cycle arrest and a proliferative block. Addition of NGI-1 to EGFR TKI treatment was synthetic lethal in cells resistant to gefitinib, erlotinib, or osimertinib. OST inhibition invariably disrupted EGFR N-linked glycosylation and reduced activation of receptors either with or without the T790M TKI resistance mutation. OST inhibition also dissociated EGFR signaling from other coexpressed receptors like MET via altered receptor compartmentalization. Translation of this approach to preclinical models was accomplished through synthesis and delivery of NGI-1 nanoparticles, confirmation of in vivo activity through molecular imaging, and demonstration of significant tumor growth delay in TKI-resistant HCC827 and H1975 xenografts. This therapeutic strategy breaks from kinase-targeted approaches and validates N-linked glycosylation as an effective target in tumors driven by glycoprotein signaling.Significance:EGFR-mutant NSCLC is incurable despite the marked sensitivity of these tumors to EGFR TKIs. These findings identify N-linked glycosylation, a posttranslational modification common to EGFR and other oncogenic signaling proteins, as an effective therapeutic target that enhances tumor responses for EGFR-mutant NSCLC. Cancer Res; 78(17); 5094-106. ©2018 AACR.

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Autophagy inhibition improves the cytotoxic effects of receptor tyrosine kinase inhibitors.

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Autophagy inhibition improves the cytotoxic effects of receptor tyrosine kinase inhibitors.

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