Prostate cancer (PCa) is the second most frequently diagnosed cancer worldwide and the fifth leading cause of cancer-related death in men. The lack of data linking specific genomic alterations to targeted treatment strategies has hindered progress in disease management. Genomic rearrangements involving the ETS transcription factors ERG or ETV1 are among the most frequent genetic alterations in PCa; however, their clinical utility remains elusive.

We used prostate-derived cellular models overexpressing ETV1 or ERG to investigate the impact of ETS overexpression on EGFR/STATs signaling by western-blot. The relevance of EGFR-STAT3 activation for ETS-driven oncogenic signaling was evaluated through targeted pharmacological inhibition using EGFR (Erlotinib) and STAT3 (TTI-101) inhibitors. Synergism studies were performed using the MTT assay and analyzed with SynergyFinder. The therapeutic potential of the Erlotinib+TTI101 combination was assessed in both 2D and 3D spheroid cultures by measuring cell viability and apoptosis using Calcein-AM/Propidium Iodide dual staining and spheroid area/size, respectively. Data were acquired and analyzed using a Cytation C10 confocal image reader.

We unveiled a positive feedback loop between ETV1 and EGFR, with STAT3 acting as a downstream effector of ETV1-EGFR signaling. Analysis of external datasets revealed that both EGFR and STAT3 are significantly upregulated in ETV1-positive PCa, consistent with ChIP-seq data identifying them as direct transcriptional ETV1 targets. Among the several concentrations tested, a promising synergistic combination of 10 µM TTI-101 with 5 µM erlotinib was identified. Accordingly, this promising combination led to a significant reduction in 2D and 3D cell growth of early and advanced PCa cells overexpressing ETV1, demonstrating a synergistic therapeutic effect and suggesting that ETV1-driven tumors may be more susceptible to this combined therapeutic approach.

Collectively, our findings highlight EGFR-STAT3 activation as a novel ETV1-regulated oncogenic pathway, providing a rationale for repurposing EGFR inhibitors in combination with STAT3 inhibitors as a therapeutic strategy for the 8-10% of prostate carcinomas characterized by ETV1 rearrangements/overexpression.