Currently, PCa is challenging the survival and quality of life of patients; its mortality rate has increased stably even with early surveillance using prostate-specific antigen (PSA). Androgen-dependent PCa (ADPC) can be effectively treated with androgen deprivation therapy (ADT). However, tumor cells can become resistant to this kind of treatment due to genomic reprogramming or other causes; in this case, ADPC evolves toward castration-resistant PCa (CRPC). In CRPC, AR mutation, overexpression and alternative splicing occurs in tumor cells, and the effect of AR signaling on tumor cell growth is significantly diminished. Furthermore, androgen can be released from tumor cells themselves or the adrenal gland. Based on this understanding, novel endocrine therapeutics have been developed that decrease androgen synthesis and the binding between androgen and AR. Abiraterone is an inhibitor of CYP17, which is crucial for androgen synthesis in testis, adrenal gland and tumor cells. After ADT treatment, tumor cells are more sensitive to low levels of androgen; hence, it is an alternative choice for effective PCa treatment that completely blocks androgen synthesis. Moreover, AR activity in tumor cells is promoted by binding androgen; therefore, agents blocking the binding of AR and androgen could show an inhibitory effect on PCa progression. Enzalutamide was designed to competitively bind AR, and tumor cell growth can be inhibited by repressing the activity of AR signaling. Generally, both abiraterone and enzalutamide were designed to target the AR signaling axis, and their efficacies for PCa inhibition were promising at the early stage. However, these inhibitory roles can be alleviated or even disappear after several months of treatment.
Therefore, the underlying mechanism for resistance or relapse needs to be further explored, and a breakthrough for effective treatment of PCa could be achieved from this point of view. PTEN and CDKN1B have been extensively demonstrated to be deleted or mutated in PCa using cohorts from Western countries and Asian populations. These findings suggest that PCa progression can be significantly inhibited by PTEN and CDKN1B overexpression. By combination with the rAAV vector, the genes of interest can be effectively delivered into normal prostate tissue and PCa cells as described previously. Furthermore, our data showed that PTEN and CDKN1B indeed inhibited tumor cell growth in vitro and in vivo, revealing an opportunity for the development of alternative treatments for PCa.
In terms of the suppressive role of PTEN, the TCGA data suggested that PTEN deletion or mutation dramatically decreased the survival of PCa patients (data not shown), and its low expression was stably detected in samples with different Gleason scores and lymph nodal metastatic status, suggesting that PTEN could be an ideal target for PCa treatment. Interestingly, the synergistic expression of PTEN and CDKN1B further hinted at the potential role of PTEN gene delivery in inhibiting PCa. Through regulating CDKN1B and CCND1 expression, PTEN can significantly inhibit the cell cycle at G1 phase and promote both early-stage and late-stage apoptosis of PCa cells in vitro.
Notably, intraprostatic injection of rAAV9.Pten and rAAV9.Cdkn1b markedly increased the survival rates of TRAMP mice. The median survival time increased from 220 days in the mock group to 301 and 365 days in the Pten and Cdkn1b groups, respectively. Single deletion of CDKN1B did not lead to tumorigenesis; however, the reduction of Cdkn1b gene expression accelerated tumorigenesis in Pten+/− mice. Conversely, the combined delivery of PTEN and CDKN1B would probably show a greater therapeutic effect on PCa than single gene delivery. Consistent with the in vitro results, PTEN promoted CDKN1B expression and CTNNB1 phosphorylation and decreased CCND1 expression in vivo, again verifying the mechanism of PTEN in regulating PCa progression. Importantly, the phosphorylation of CTNNB1 is directly regulated by PTEN/AKT/GSK3β signaling pathway, namely, PTEN overexpression activates GSK3β function and increases CTNNB1 phosphorylation, and then the phosphorylation of CTNNB1 was degraded by proteasome[32, 33]. Therefore, the expression of its downstream genes promoting cell cycle and survival was decreased, and the cancer progression was further inhibited.
In our study, 4 × 1011 GC rAAVs were administered intraperitoneally amounting to 1.74 × 1013 GC/kg, based on an average weight of 8-weeks old mice of 23 g. Although, this dose appears to be high, a preclinical study in dogs using semisystemic intraportal administration of rAAV8 reported the safe administration of 4.95 × 1013 GC/kg, supporting the safety and feasibility of our dose. In particular, our study employed two in vivo mouse models to investigate the therapeutic role of PTEN and CDKN1B in PCa. The TRAMP mouse model simulated the PCa condition by exhibiting various forms of disease from mild intraepithelial hyperplasia to large multinodular malignant neoplasia. The TRAMP-C2 xenograft model was obtained by injecting TRAMP-C2 cells into wild-type C57/B6 mice. These two models were established with a normal immune environment; hence, the therapeutic effect of rAAV-based gene delivery, demonstrated by its inhibition of PCa progression, provides strong evidence for its clinical transformation and application.