Elucidation of Ras homologous A: Systematic approach to identify potential signaling in gastric cancer

Previously, we identified Ras homologous A (RHOA) as a major signalling hub in gastric cancer (GC), the third-most common cause of cancer death in the world, prompting us to rationally design an efficacious inhibitor of this oncogenic GTPase. Here, based on that previous work, we extend those computational analyses, and in silico modeling approaches, to further pharmacologically optimize anti-RHOA hydrazide derivatives for greater anti-GC potency. Two of these, JK-136 and JK-139, potently inhibited cell viability and migration/invasion of GC cell lines, and mouse xenografts, diversely expressing RHOA. Moreover, JK-136’s binding affinity for RHOA was >140-fold greater than Rhosin, a nonclinical RHOA inhibitor. Network analysis of JK-136/139-associated transcriptomes showed different functional contexts, compared to those following treatment with Rhosin. We strongly assert that identifying and targeting oncogenic signalling hubs, such as RHOA, represents an emerging strategy for the design, characterization, and translation of new antineoplastics, against gastric and other cancers. The Affymetrix Whole transcript Expression array process was executed according to the manufacturer's protocol (GeneChip Whole Transcript PLUS reagent Kit). cDNA was synthesized using the GeneChip WT (Whole Transcript) Amplification kit as described by the manufacturer. The sense cDNA was then fragmented and biotin-labeled with TdT (terminal deoxynucleotidyl transferase) using the GeneChip WT Terminal labeling kit. Approximately 5.5 μg of labeled DNA target was hybridized to the Affymetrix GeneChip Human 2.0 ST Array at 45°C for 16hour. Hybridized arrays were washed and stained on a GeneChip Fluidics Station 450 and scanned on a GCS3000 Scanner (Affymetrix). Signal values were computed using the Affymetrix® GeneChip™ Command Console software.