NSCLC patients undergoing platin chemotherapy often develop resistance within 9-12 months and drug-resistance is the primary reason for the low median survival of 10-14 months among stage IV patients. Therefore, resistance is the fundamental therapeutic limitation in the later part of the progressive disease. Researchers showed that suppressed Caspase-3 is one of the primary reasons for failure to induce apoptosis by the cell during cisplatin resistance. Recently, AXL and TWEAK/FN14 pathways have been shown to be upregulated in cisplatin-resistant cells causing activation of EMT and survival pathways. Therefore, we examined whether targeted dual inhibition of AXL and FN14 successfully reverses the cisplatin resistance in NSCLC.
We tested cisplatin-resistant EGFR mutant H1975 and KRAS mutant A549 cells following knockdown of AXL and FN14. We used gelatin nanoparticles conjugated(gelNP) with AXL and FN14 siRNAs and cetuximab as a targeting agent. We compared efficacy of gelNP with small molecule AXL and FN14 pharmacological inhibitors. Mice (n = 27) were divided into four groups and treated with cisplatin, AXL/FN14 inhibitors, and gelNP along with controls. We investigated cellular toxicity(IC50) using MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye assay and mechanism using western blot. Cytochrome-C induced apoptosis was detected using fluorescence microscopy.
There was a marked decrease in average tumor volume, both in pharmacological and genetic inhibitor (gelNP) treated groups. IC50 values decreased significantly in dual inhibition (<200%). Mutual regulation of AXL and FN14 were observed, presumed through src-RhoA-ROCK pathways. Although in-vitro AXL suppression showed increase in EGFR, dual inhibition reversed the trend. During dual inhibition decreased p-src, p-50, p-AKT, and survivin, and increased FHIT, cleaved caspase 3 and cleaved PARP levels were observed. Cytochrome c release further confirmed intrinsic apoptosis.
Our results show that targeted dual inhibition of AXL and TWEAK/FN14 using targeted therapy by gelNP can reverse cisplatin resistance by inducing higher caspase 3 cleavage through FHIT upregulation that can enhance survival of NSCLC patients undergoing chemotherapy.
University of Missouri-Columbia, USA
Michael J and Sharon R Bukstein Endowment funds
All authors have declared no conflicts of interest.