Enzalutamide resistance in LNCaP due to dysregulation of the retinoblastoma-E2F complex

INTRODUCTION Enzalutamide (Enz) is a recently FDA-approved anti-androgen used for treatment of metastatic, advanced prostate cancer. As learnt from first-generation anti-androgens, therapy with Enz will eventually lead to therapy-resistance. Understanding the mechanism responsible for Enz-resistance will provide useful in defining new therapeutic targets and predicting Enz-response in patients. An in vitro model for Enz-resistance was generated by long-term therapy of LNCaP cells with 10 µM Enz. Proliferation of the resistant LNCaP cells, named LNCaP-Rr, was no longer repressed by Enz. METHODS and RESULTS The LNCaP-Rr cells were investigated for transcriptomic changes via RNA-seq analysis. For a large set of androgen-regulated genes in LNCaP, we observed a weakened androgen response in the LNCaP-Rr, especially for the PSA gene. This lower AR activity could not be attributed to lower levels of AR protein nor by an acquired somatic mutation in the AR gene. Besides the LNCaP-specific T877A mutation, no additional mutations were observed in the LNCaP-Rr after sequencing of the AR. Striking changes were observed in the androgen-regulated expression of genes involved in cell cycle progression. While there was no prominent change in the mRNA level of RB1, a clear change in the mRNA expression of E2F-transcription factors was observed. Moreover, almost 50% of E2F target genes were affected in a similar way. Functionally, the affected genes are involved in cell cycle, DNA synthesis, replication and DNA damage repair. Further study at the protein level revealed high levels of RB1 in the resistant cells but unaffected levels of E2F1 and E2F2. A changed control of RB1 on the activity of the E2F transcription factors is therefore suggested as an underlying cause for Enz-resistance. In search of alternative therapies after establishment of Enz-resistance, several agents were tested for their antiproliferative effects on LNCaP-Rr. The LNCaP-Rr cell line has developed cross resistance to docetaxel, a chemotherapeutic agent. Moreover, the cell line is partly resistant to PD-0332991, a highly selective CDK4/6 inhibitor, and to MEL6, an in-house AR antagonist. The response of LNCaP-Rr to galeterone and abiraterone, which are steroidal CYP17A1 inhibitors and AR antagonists, however, remained unaffected. In analogy with the LNCaP-Rr cells, these data suggest that in some Enz-resistant patients, the AR can still be targeted by some specific, structurally different AR antagonists but that the use of chemotherapy has to be reconsidered.status: publishe