Elda Grabocka

Supplementary Figure from Stress Granules Determine the Development of Obesity-Associated Pancreatic Cancer

Supplementary Data from Stress Granules Determine the Development of Obesity-Associated Pancreatic Cancer

Figure S5 shows Casp3 levels through cell cycle phases, protein levels of full length and cleaved CASPASE 9, CASPASE 2, PARP-1, and fraction of live, dead, and apoptotic cells in asynchronous and G2-phase synchronized PDAC cells, quantification of full length and cleaved cPLA2 and CASPASE-3 in asynchronous and G2-phase synchronized PDAC cells, expression of wild type cPLA2 and the CASPASE-3-cleavage resistant cPLA2 mutant in endogenous cPLA2 knockdown human PDAC cells and their impact of 15d-PGJ2 levels and SG formation.

Figure S2 shows the impact of 15d-PGJ2 on the cell cycle profile of PDAC cells, COX2 and HPGD in the context of the arachidonic acid pathway and KRAS, the levels of COX2 and HPGD in asynchronous and G2-phase PDAC cells, intercellular heterogeneity of SGs and G2-phase upregulation of SGs in normal cells, and the levels of L-PGDS in asynchronous and G2-phase PDAC cells.

Figure S6 shows drug treatment schematics, impact of cPLA2 inhibition on SG formation in Geminin positive (S/G2-phase) and negative (G1-phase) in murine PDAC, impact of gemcitabine and oxaliplatin on the cell cycle profile of murine PDAC cells, and effect of cPLA2 inhibition on murine PDAC sensitivity to gemcitabine.

Figure S3 shows the levels of arachidonic acid and activity of PLC, PLD, and PLA2 in asynchronous and G2-phase synchronized PDAC cells, effect of cPLA2-i on SG formation in asynchronous and G2-phase synchronized normal cells, effects of sPLA2-i and iPLA2-i on SG formation in asynchronous and G2-phase synchronized PDAC cells, impact of PLC, PLD, cPLA2, iPLA2, and cPLA2 inhibitors on cell cycle distribution and 15d-PGJ2 levels in asynchronous and G2-phase synchronized PDAC cells, and specific inhibition of SG formation in G2-phase by cPLA2 knockdown.

Figure S1 shows intercellular heterogeneity of SGs under various types of stress, cell cycle distribution of PDAC cells, SG formation across cell cycle phases determined by: FastFUCCI, mitotic morphology, FACS-based DNA content following synchronization by double thymidine or the CDK1 inhibitor Ro-3306, time lapse analysis of SG formation in G2-phase synchronized and asynchronous PDAC cells expressing the SG marker G3BP1 fused to GFP (G3BP1-GFP), and quantification of SG formation via multiple markers of SGs in asynchronous and G2-phase synchronized PDAC cells.

Figure S4 shows quantifications of the unphosphorylated and phosphorylated levels of the full-length (FL) and truncated (TR) cPLA2 proteins across FastFUCCI sorted G1-, S-, and G2-phase PDAC cells, transcript levels of PLA2G4A in asynchronous and G2-phase synchronized PDAC cells, protein expression levels of PLC and PLD in asynchronous and G2-phase synchronized PDAC cells, protein expression levels of inducible TR-cPLA2, and reversal of SGs suppression by induced expression of TR-cPLA2 in G2-phase synchronized PDAC cells through administration of exogenous 15d-PGJ2.

Cancer cell heterogeneity is a major therapeutic challenge. In this study, we identify that individual cells within cancer cell populations show significant heterogeneity in the levels of the stress-adaptive organelles, stress granules (SG), and demonstrate that SG heterogeneity is dictated by the cell cycle state. Specifically, SG formation is distinctively heightened in cells in the G2 phase because of the interplay between a nonapoptotic function of CASPASE-3 and calcium-dependent phospholipase A2 (cPLA2)–mediated production of the SG-promoting molecule, 15-deoxy-delta-prostaglandin-J2. We demonstrate that in the G1–S phase, CASPASE-3 cleaves and inactivates cPLA2, whereas in the G2 phase, CASPASE-3 activity is suppressed, resulting in enhanced cPLA2 activity and 15-deoxy-delta-prostaglandin-J2 upregulation. We show that cell cycle–dependent SG heterogeneity is a property of pancreatic ductal adenocarcinoma and targeting G2-SGs by inhibiting cPLA2 sensitizes pancreatic ductal adenocarcinoma to G2 arrest–inducing chemotherapeutics. Our findings highlight cell cycle–dependent SG formation as a fundamental property of SGs, a key aspect of cancer heterogeneity, and a target for cancer treatment.Significance:Because of their defective G1 checkpoint mechanisms, cancer cells often activate a G2 checkpoint, which leads to resistance to DNA-damaging chemotherapeutics. We identify an intercellular heterogeneity of SGs that is driven by the cell cycle, with SG formation being highest in the G2 phase. Targeting G2-specific SG formation sensitizes pancreatic tumors to G2 arrest–inducing chemotherapeutics.

Biomolecular condensates partition various cellular processes including transcription, DNA repair, and RNA metabolism. We report NELFE, a member of the Negative Elongation Factor complex required for Polymerase II (Pol II) pausing, forms distinct foci mediated by two low complexity sequences. We show NELFE is oncogenic in hepatocellular carcinoma (HCC) by undergoing liquid-liquid phase separation (LLPS) with SMARCB1 to modulate chromatin accessibility to downregulate pro-apoptotic genes through Pol II pausing while activating pro-growth signals to promote HCC progression. Our work highlights the importance of NELFE LLPS as a mechanism of chromatin accessibility to regulate both paused and non-paused genes to drive tumorigenesis in hepatocellular carcinoma.