Targeting the DNA Double Strand Break Repair Machinery in Prostate Cancer

Regardless of the achievable remissions with first line hormone therapy in patients with prostate cancer (CaP), the disease escapes the hormone dependent stage to a more aggressive status where chemotherapy is the only effective treatment and no treatment is curative. This makes it very important to identify new targets that can improve the outcome of treatment. ATM and DNA-PK are the two kinases responsible for signalling and repairing double strand breaks (DSB). Thus, both kinases are pertinent targets in CaP treatment to enhance the activity of the numerous DNA DSB inducing agents used in CaP treatment such as ionizing radiation (IR). Colony formation assay was used to assess the sensitivity of hormone dependent, p53 wt (LNCaP) and hormone independent p53 mutant (PC3) CaP cell lines to the cytotoxic effect of IR and Doxorubicin in the presence or absence of Ku55933 and NU7441 which are small molecule inhibitors of ATM and DNA-PK, respectively. Flow cytometry based methods were used to assess the effect of the two inhibitors on cell cycle, apoptosis and H2AX foci formation. Neutral comet assay was used to assess the induction of DNA DSBs. Ku55933 or NU7441 alone increased the sensitivity of CaP cell lines to the DNA damaging agents, however combining both inhibitors together resulted in further enhancement of sensitivity. The cell cycle profile of both cell lines was altered with increased cell death, DNA DSBs and H2AX foci formation. This study justifies further evaluation of the ATM and DNA-PK inhibitors for clinical application in CaP patients. Additionally, the augmented effect resulting from combining both inhibitors may have a significant implication for the treatment of CaP patients who have a defect in one of the two DSB repair pathways

Regulation of the androgen receptor by SET9-mediated methylation

The androgen receptor (AR) is a member of the nuclear hormone receptor family of transcription factors that plays a critical role in regulating expression of genes involved in prostate development and transformation. Upon hormone binding, the AR associates with numerous co-regulator proteins that regulate the activation status of target genes via flux to the post-translational modification status of histones and the receptor. Here we show that the AR interacts with and is directly methylated by the histone methyltransferase enzyme SET9. Methylation of the AR on lysine 632 is necessary for enhancing transcriptional activity of the receptor by facilitating both inter-domain communication between the N- and C-termini and recruitment to androgen-target genes. We also show that SET9 is pro-proliferative and anti-apoptotic in prostate cancer cells and demonstrates up-regulated nuclear expression in prostate cancer tissue. In all, our date indicate a new mechanism of AR regulation that may be therapeutically exploitable for prostate cancer treatment

Nutlin-3 inhibits androgen receptor-driven c-FLIP expression, resulting in apoptosis of prostate cancer cells

Inhibition of androgen receptor (AR) signalling represents the conventional medical management of prostate cancer. Ultimately this treatment fails because tumors develop an incurable, castrate resistant phenotype, resulting in an unmet need for new treatments in prostate cancer. The AR remains a viable therapeutic target in castrate resistant disease, such that novel ways of downregulating AR activities are attractive as potential treatments. Here we describe a mechanism by which the AR can be downregulated by the MDM2 antagonist Nutlin-3, resulting in loss of pro-survival c-FLIP gene expression and apoptosis. We additionally show that loss of c-FLIP sensitises prostate cancer cells to Nutlin-3. Finally, we demonstrate that the unrelated MDM2 antagonist Mi-63 also impinges upon AR signalling, supporting the concept of future treatment of prostate cancer with MDM2 antagonists

Targeting the DNA double strand break repair machinery in prostate cancer

Prostate cancer is the most common cancer in males in western societies. In spite of the successful first line treatment using surgery, radiation therapy, antiandrogen treatment or combination therapy the disease progresses towards a hormone refractory state where the only effective treatment is chemotherapy which prolongs overall survival, however it is not curative. The resistance that hormone refractory disease displays highlights the importance of developing new targeted therapies which may be curative or at least may improve the patient's quality of life and overall survival. Current chemotherapeutic regimens used in prostate cancer treatment mostly contain agents that induce DNA damage and specifically double strand breaks, such as Doxorubicin, mitoxantrone and etoposide.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Ku55933 and NU7441 increased DNA DSB in CaP cell lines.

<p><b>LNCaP (A&C)</b> and <b>PC3 (B&D)</b> cells were treated with 10 Gy or 1 µM doxorubicin following 1 h incubation with Ku55933 (10 µM) and/or NU7441 (1 µM) and stained with FITC-conjugated H2AX antibody before being analysed directly on FACscan. Results are the mean of at least 5 independent experiments±SEM. <b>PC3 (E)</b> cells were treated as above for 24 h and analysed using comet assay, 50 cells were counted per arm treatment. Results are the mean of 3 independent experiments ± SEM.</p

Ku55933 and/or NU7441 sensitised CaP cells to IR or doxorubicin.

<p><b>LNCaP (A&B)</b> or <b>PC3 (C&D)</b> cells were seeded in different densities and left to attach before 1 h incubation with varying concentration of Ku55933 or NU7441 prior to irradiation 1 Gy or 10 nM doxorubicin treatment for 24 h (<i>P</i><0.001). <b>LNCaP (E&F)</b> or <b>PC3 (G&H)</b> cells were seeded in different densities and left to attach before 1 h incubation with Ku55933 (10 µM) or NU7441 (1 µM) prior to treatment with varying IR doses or doxorubicin concentration for 24 h (<i>P</i><0.001). All results are the mean of three independent experiments ± SEM.</p

Ku55933 and NU7441 altered CaP cell lines cell cycle and increased apoptosis.

<p><b>LNCaP (A&C)</b> and <b>PC3 (B&D)</b> cells were incubated with Ku55933 (10 µM) and/or NU7441 (1 µM) before being exposed to the indicated doses of IR or doxorubicin for 48 h and then stained with PI and analysed directly on FACscan. Data is a representative three independent experiments. <b>LNCaP (E)</b> and <b>PC3 (F)</b> cells were treated as described above and stained with FITC-conjugated anti caspase 3 antibody before direct analysis on FACScan. Results are the mean of three independent experiments ± SD.</p