Neratinib and entinostat combine to rapidly reduce the expression of K-RAS, N-RAS, Gα(q) and Gα(11) and kill uveal melanoma cells
| Autor: | Andrew Poklepovic, Cindy Sander, John M. Kirkwood, John F. Hancock, Paul Dent, Alshad S. Lalani, Laurence Booth, Jane L. Roberts |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
MAPK/ERK pathway Uveal Neoplasms Cancer Research Time Factors Pyridines Down-Regulation GTP Phosphohydrolases Proto-Oncogene Proteins p21(ras) 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Cell Line Tumor Antineoplastic Combined Chemotherapy Protocols medicine Autophagy Humans Protein kinase A Melanoma PI3K/AKT/mTOR pathway Pharmacology biology Kinase Chemistry Entinostat Membrane Proteins Drug Synergism GTP-Binding Protein alpha Subunits Gene Expression Regulation Neoplastic 030104 developmental biology Oncology 030220 oncology & carcinogenesis Mitogen-activated protein kinase Neratinib Benzamides biology.protein Cancer research Quinolines Molecular Medicine GTP-Binding Protein alpha Subunits Gq-G11 Drug Screening Assays Antitumor Janus kinase medicine.drug Research Paper |
| Popis: | There is no efficacious standard of care therapy for uveal melanoma. Unlike cutaneous disease, uveal melanoma does not exhibit RAS mutations but instead contains mutations with ~90% penetrance in either Gα(q) or Gα(11). Previously we demonstrated that neratinib caused ERBB1/2/4 and RAS internalization into autolysosomes which resulted in their proteolytic degradation. In PDX isolates of uveal melanoma, neratinib caused the internalization and degradation of Gα(q) and Gα(11) in parallel with ERBB1 breakdown. These effects were enhanced by the HDAC inhibitor entinostat. Similar data were obtained using GFP/RFP tagged forms of K-RAS V12. Down regulation of Gα(q) and Gα(11) expression and RAS-GFP/RFP fluorescence required Beclin1 and ATG5. The [neratinib + entinostat] combination engaged multiple pathways to mediate killing. One was from ROS-dependent activation of ATM via AMPK-ULK1-ATG13-Beclin1/ATG5. Another pathway was from CD95 via caspase 8-RIP1/RIP3. A third was from reduced expression of HSP70, HSP90, HDAC6 and phosphorylation of eIF2α. Downstream of the mitochondrion both caspase 9 and AIF played roles in tumor cell execution. Knock down of ATM/AMPK/ULK-1 prevented ATG13 phosphorylation and degradation of RAS and Gα proteins. Over-expression of activated mTOR prevented ATG13 phosphorylation and suppressed killing. Knock down of eIF2α maintained BCL-XL and MCL-1 expression. Within 6h, [neratinib + entinostat] reduced the expression of the immunology biomarkers PD-L1, ODC, IDO-1 and enhanced MHCA levels. Our data demonstrate that [neratinib + entinostat] down-regulates oncogenic RAS and the two key oncogenic drivers present in most uveal melanoma patients and causes a multifactorial form of killing via mitochondrial dysfunction and toxic autophagy. Abbreviations: ERK: extracellular regulated kinase; PI3K: phosphatidyl inositol 3 kinase; ca: constitutively active; dn: dominant negative; ER: endoplasmic reticulum; AIF: apoptosis inducing factor; AMPK: AMP-dependent protein kinase; mTOR: mammalian target of rapamycin; JAK: Janus Kinase; STAT: Signal Transducers and Activators of Transcription; MAPK: mitogen activated protein kinase; PTEN: phosphatase and tensin homologue on chromosome ten; ROS: reactive oxygen species; CMV: empty vector plasmid or virus; si: small interfering; SCR: scrambled; IP: immunoprecipitation; VEH: vehicle; HDAC: histone deacetylase. |
| Databáze: | OpenAIRE |
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