PO-035 LKB1 deficiency renders non-small-cell lung cancer cells sensitive to ERK inhibitor

Abstract

IntroductionThe transcriptional factor NF-κB, composed by five subunits (RelA/p65, c-Rel, RelB, p50, p52), is largely involved in many facets of cellular physiology such as innate and adaptive immunity as well as inflammation. In addition, NF-kB play a central role in cancer cell survival and chemoresistance partly by its implication in cross-talks with redox-regulating proteins. Ferritin is the major iron storage protein; it is composed by a variable assembly of Heavy (FHC) and Light (FLC) subunits. FHC, in particular, has been widely demonstrated to be devoted in iron uptake and release thus controlling the redox homeostasis.Material and methodsK562 erythroleukemia cells were stably silenced for FHC by using the shRNA method. Then FHC reconstitution was achieved by transient transfection of a FHC specific expression vector. ROS were determined by incubating cells with the redox-sensitive probe 2‘−7‘-DCF. NAC was used to inhibit ROS production. MTT assay was performed to analyse cell viability. Increasing concentrations of Doxorubicin, ranging from 0 to 5 µM, were used to treat K562 cells.Results and discussionsThe results of this study highlighted that FHC amounts negatively affect NF-kB activation in K562 cells. FHC silencing was accompanied by an increased expression of the nuclear NF-kB subunit p65, FHC rescue determined nuclear p65 decrease. FHC silencing is responsible for intracellular ROS production and ROS are implicated in NF-kB pathway. To elucidate the relationship between ROS amount and nuclear p65 content, we determined ROS amounts in our in vitro model and evaluated p65 nuclear expression after treatment with the ROS scavenger NAC. First, we observed that, as expected, ROS levels increased upon FHC silencing and return to basal levels upon NAC treatment. Interestingly, NAC was also able to decrease nuclear p65 amount in FHC-silenced K562 cells. Considering the effect of NF-kB activated pathway on cell survival, we analysed the effect of FHC silencing-mediated p65 increase in K562 cells upon treatment with increasing doses of Doxorubicin. Cell viability assay highlighted that FHC-silencing was accompanied by an increased resistance to the drug with an IC50 about doubled compared to that of the K562 control cells at each the time points. This resistance of FHC-silenced cells was reverted upon NF-kB inhibitor transfection.ConclusionFHC silencing induced NF-kB activation in K562 cells through the modulation of intracellular ROS content. This regulatory axis can be used to modulate K562 chemoresistance.