Combination Therapeutic Strategies Targeting Growth and Metabolic Pathways in Prostate Cancer

Abstract

Despite recent advances, prognosis in metastatic prostate cancer remains poor. As with other cancers, tumor heterogeneity is an increasingly evident contributor in prostate tumorigenesis and developed resistance. Using in vitro and in vivo model systems, we examined novel diagnostic and therapeutic strategies in prostate cancer. In these studies, combination treatment with amuvatinib, a receptor tyrosine kinase inhibitor, and erlotinib, an epidermal growth factor inhibitor, was assessed for its ability to differentially modulate growth signaling in pathway diverse LNCaP (PTEN⁻) and DU-145 (PTEN⁺) human prostate cancer cell and mouse xenograft models. Our results suggest both individual mechanistic signaling activities, as well as benefits of the combination therapy though modulations of MAPK (pERK) and 4EBP1/cyclin D1 in growth signaling divergent PTEN+ and PTEN- prostate cancer cells. Additionally, despite the importance preanalytical tissue preservation on downstream diagnostic assays, exact protocols are not well defined and highly variable clinically and, as such, critical diagnostic information is lost. We show that a novel 2+2 fixation method induces target- and cell-specific alterations in immunostain intensity and efficacy. Importantly, cyclin D1 is increasingly utilized for as a clinical prognostic/diagnostic marker and demonstrated improved immunohistochemical staining efficacy with 2+2 fixation compared with treatment-matched xenograft protein alterations as assessed by western analysis. Finally, pentoxifylline (PTX) is a clinically utilized and well tolerated PDE inhibitor that has shown promise as a radio-/chemo-sensitization and anti-cancer agent against a variety of cancers. In these studies, we demonstrate that PTX induces cell and tumor growth inhibition in LNCaP prostate cancer cells. Mechanistically, PTX induces transient cellular signaling modulations of both the AMPK metabolic and AKT/mTOR growth pathways, while inducing autophagy. Also, PTX sensitizes LNCaP prostate cancer to cytotoxicity induced by first line chemotherapy docetaxel, inducing significant cellular apoptosis and reducing effective docetaxel concentrations by >10 fold for equivalent toxicity in viability assays. These findings nominate PTX as an adjunct therapy for the treatment of prostate cancer. In summary, these studies characterize the targeted signaling modulation by combination erlotinib and amuvatinib therapy, as well as pentoxifylline, for their use as therapies for prostate cancer. A novel fixation protocol was also assessed for improved diagnostic tissue preservation of critical signaling proteins. Further understanding in these areas will aid and expand the development of effective diagnostics, as well as emphasize the benefits of these and similar therapeutics for the treatment of prostate cancer.