Localized prostate cancer (PCa) is often curable, whereas metastatic disease treated by castration inevitably progresses toward castration-resistant PCa (CRPC). Most CRPC treatments target androgen receptor (AR) signaling. However, not all CRPC cells rely on AR activity for survival and proliferation. With advances in immunotherapy and fluid biopsies for cancer management, expression systems specific for both AR-positive and -negative PCa are required for virus-based vaccines and cell imaging. To target both AR-responsive and non-responsive cells, we developed a three-step transcriptional amplification (3STA) system based on the progression elevated gene-3 ( ) promoter named -3STA. Notably, we report on different genetic modifications that significantly improved PEG3 promoter's strength in PCa cells. Adenoviruses incorporating promoter with and without transcriptional amplification systems were generated. The potential of -3STA to target PCa cells was then evaluated in vitro and in vivo in androgen-responsive and non-responsive PCa cell lines. -3STA was shown to be active in all PCa cell lines and not regulated by androgens, and its activity was amplified 97-fold compared to that of a non-amplified promoter. The -3STA system can thus be used to target advanced AR+ and AR- cells for imaging or immunovirotherapy in advanced PCa.