GSE158569
Targeting tumor-macrophage interaction via the Notch2-Jag1 axis reverses tumor resistance to paclitaxel
Taxanes are widely used in chemotherapy, but intrinsic and acquired resistance limit the clinical outcomes. Studies showed tumor interaction with suppressive macrophages plays a key role in taxane resistance, yet therapeutic strategies that deplete or repolarize macrophages are challenging. Here we uncovered a novel tumor-macrophage interaction via Notch2-Jag1 justacrine signaling that can be targeted to sensitize paclitaxel response without affecting the broad macrophage functions. Using translatome profiling, we identified Notch2 upregulation during taxol-induced prolonged mitosis. Notch2 was subsequently activated in the post-mitotic G1 phase by Jag1 expressed on neighboring macrophages, which promoted tumor cell survival by upregulating p38 and anti-apoptotic proteins. Notch2 also upregulated cytokines that further recruited Jag1-expressing macrophages. By targeting this Notch2-Jag1 interaction with a pan-Notch inhibitor, RO4929097, taxol resistance was significantly attenuated in multiple mouse tumor models. Our results point to combining Notch inhibitor with taxane as an effective strategy to selectively disrupt tumor-macrophage interaction underlying chemoresistance. Overall design: For ribosome profiling, HeLa cells were synchronized by double thymidine (2 mM, Sigma). Then the dishes were washed three times with PBS to release the cells in complete DMEM again. 9h later, DMSO and 5nM Taxol were added to each group, respectively. The mitosis cells were collected by shake-off. And the mitosis cells from DMSO and Taxol treatment were defined as mitosis(M) and prolonged mitosis(pM) cells, respectively. Mitotic (M) and prolonged mitotic (pM) HeLa cells were lysed in lysis buffer (20 mM Tris pH 7.5, 150 mM KCl, 5 mM MgCl2, 1 mM dithiothreitol, 8% glycerol) supplemented with 0.5% Triton X-100, 30 U/ml Turbo DNase (Ambion, Life Technologies, CA, United States) and 100 µg/ml cycloheximide (Sigma Aldrich, MO, United States). Ribosome protected fragments were then isolated and sequenced as previously described (PMID: 22056041). The resulting mRNAs were modestly fragmented by partial hydrolysis in bicarbonate buffer so that the average size of fragments was ~80 bp. The fragmented mRNAs were separated by denaturing PAGE and fragments of 26–34 nt were selected. The sequencing libraries were prepared and sequenced as previously described (PMID: 22056041).The differential gene translation profile between M and pM were analyzed. The differential gene expression profile between wild type and Notch2 knockdown OVCAR8 cell lines were analyzed. RNA was extracted from OVCAR8 cells using Trizol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. Subsequently, total RNA was quantified using a Nano Drop and Agilent 2100 bioanalyzer (Thermo Fisher Scientific, MA, USA). The Beijing Genomics Institute (BGI, Shenzhen, China) performed the RNA integrity checking, library preparation and sequencing.Each group was performed in two replicates. Notch2 knockdown OVCAR8 cells were established using plko.1 lentivirus system and two shRNAs target different sequences on Notch2 mRNA were used. Subsequently, we confirmed the RNAseq result in ovarian cancer cells by qPCR (data not included).