Aberrant translation initiation at non-AUG start codons is associated with multiple cancers and neurodegenerative diseases. Nevertheless, how non-AUG translation may be regulated differently from canonical translation is poorly understood. Here, we used start codon-specific reporters and ribosome profiling to characterize how translation from non-AUG start codons responds to protein synthesis inhibitors in human cells. These analyses surprisingly revealed that translation of multiple non-AUG-encoded reporters and the endogenous GUG-encoded DAP5 (eIF4G2/p97) mRNA is resistant to cycloheximide (CHX), a translation inhibitor that severely slows but does not completely abrogate elongation. Our data suggest that slowly elongating ribosomes can lead to queuing/stacking of scanning preinitiation complexes (PICs), preferentially enhancing recognition of weak non-AUG start codons. Consistent with this model, limiting PIC formation or scanning sensitizes non-AUG translation to CHX. We further found that non-AUG translation is resistant to other inhibitors that target ribosomes within the coding sequence but not those targeting newly initiated ribosomes. Together, these data indicate that ribosome queuing enables mRNAs with poor initiation context-namely, those with non-AUG start codons-to be resistant to pharmacological translation inhibitors at concentrations that robustly inhibit global translation.

Minimally invasive ablation strategies enable locoregional treatment of tumors. One such strategy, electrolytic ablation, functions through the local delivery of direct current without thermal effects, facilitating enhanced precision. However, the clinical application of electrolytic ablation is limited by an incompletely characterized mechanism of action. Here we show that acid and base production at the electrodes precipitates local pH changes causing the rapid cell death that underlies macroscopic tumor necrosis at pH > 10.6 or < 4.8. The extent of cell death can be modulated by altering the local buffering capacity and antioxidant availability. These data demonstrate that electrolytic ablation is distinguished from other ablation strategies via its ability to induce cellular necrosis by directly altering the tumor microenvironment. These findings may enable further development of electrolytic ablation as a curative therapy for primary, early stage tumors.

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Background: Locoregional therapy is playing an increasingly central role in the management of hepatocellular carcinoma (HCC), where ablation is distinguished among these techniques by its capacity to effect cure. Electrochemical treatment (EChT), a technique that facilitates necrosis via the application of direct current at low voltages shows promise; however, widespread use has been limited as studies of the underlying biology and the mechanism of action have been limited. Methods: A novel assay for the study of EChT mechanism was designed, wherein HCC cells (2.5e5 cells/mL) were embedded in low-melting temperature agarose (1.5%). EChT was performed on these assays between nitinol cathodes (d = 0.5 mm) and platinum anodes (d = 0.5 mm) in a variety of geometries. Buffering capacity of the encapsulation assay was varied via the addition of HEPES buffer at final concentrations of 10mM, 50mM, or 200mM. Either during or immediately following EChT, the following measurements were recorded: cell viability, pH, ROS burden, transmembrane potential, and temperature. Results: Following EChT, acidic pH was appreciated surrounding the anodes and basic pH surrounding the cathodes in regions where cell death was observed by fluorescence microscopy. Upon increasing the buffering capacity of the assay, the total area of cell death decreased (p &lt; 10-6). There was not sufficient heat generation nor transmembrane potential to account for the observed cell death. A pH-threshold was identified, where a pH &gt; 10.8 or &lt; 4.4 was found to cause cell necrosis in the three different buffer conditions studied. By varying the number of electrodes, their spacing, and length of ablation, it was possible to shape the zone of cell death observed to an arbitrary geometry. Conclusions: The mechanism of EChT-induced cancer cell death is through the spread of acidic and basic species generated by hydrolysis of water upon electron transfer at the electrodes. The diffusion of these species leads to cell death in a predictable, pH dependent manner. These results suggest that an electrochemical ablation device could treat complex HCC tumor geometries with appropriate electrode placement and charge delivery. Citation Format: Nicholas Perkons, Elliot Stein, Chike Nwaezeapu, Joseph Wildenberg, Daniel Ackerman, Gregory Nadolski, Stephen Hunt, Terence Gade. Electrochemical treatment produces pH changes in the tumor microenvironment that are toxic to cancer cells [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 195.